Compounds that modulate intracellular calcium

ABSTRACT

Described herein are compounds and pharmaceutical compositions containing such compounds, which modulate the activity of store-operated calcium (SOC) channels. Also described herein are methods of using such SOC channel modulators, alone and in combination with other compounds, for treating diseases or conditions that would benefit from inhibition of SOC channel activity.

CROSS-REFERENCE

This application is a divisional patent application of U.S. patentapplication Ser. No. 12/192,812, entitled “COMPOUNDS THAT MODULATEINTRACELLULAR CALCIUM” filed Aug. 15, 2008, which claims the benefit ofU.S. provisional application Ser. No. 60/971,161, entitled, “CompoundsThat Modulate Intracellular Calcium” filed Sep. 10, 2007, all of whichare herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

Described herein are compounds, pharmaceutical compositions andmedicaments that include such compounds, and methods of using suchcompounds to modulate store operated calcium (SOC) channel activity.

BACKGROUND OF THE INVENTION

Calcium plays a vital role in cell function and survival. For example,calcium is a key element in the transduction of signals into and withincells. Cellular responses to growth factors, neurotransmitters, hormonesand a variety of other signal molecules are initiated throughcalcium-dependent processes.

Virtually all cell types depend in some manner upon the generation ofcytoplasmic Ca²⁺ signals to regulate cell function, or to triggerspecific responses. Cytosolic Ca²⁺ signals control a wide array ofcellular functions ranging from short-term responses such as contractionand secretion to longer-term regulation of cell growth andproliferation. Usually, these signals involve some combination ofrelease of Ca²⁺ from intracellular stores, such as the endoplasmicreticulum (ER), and influx of Ca²⁺ across the plasma membrane. In oneexample, cell activation begins with an agonist binding to a surfacemembrane receptor, coupled to phospholipase C (PLC) through a G-proteinmechanism. PLC activation leads to the production of inositol1,4,5-triphosphate (IP₃), which in turn activates the IP₃ receptorcausing release of Ca²⁺ from the ER. The fall in ER Ca²⁺ then signals toplasma membrane store-operated calcium (SOC) channels.

Store-operated calcium (SOC) influx is a process in cellular physiologythat controls such diverse functions such as, but not limited to,refilling of intracellular Ca²⁺ stores (Putney et al. Cell, 75, 199-201,1993), activation of enzymatic activity (Fagan et al., J. Biol. Chem.275:26530-26537, 2000), gene transcription (Lewis, Annu. Rev. Immunol.19:497-521, 2001), cell proliferation (Nunez et al., J. Physiol. 571.1,57-73, 2006), and release of cytokines (Winslow et al., Curr. Opin.Immunol. 15:299-307, 2003). In some nonexcitable cells, e.g., bloodcells, immune cells, hematopoietic cells, T lymphocytes and mast cells,SOC influx occurs through calcium release-activated calcium (CRAC)channels, a type of SOC channel.

The calcium influx mechanism has been referred to as store-operatedcalcium entry (SOCE). Stromal interaction molecule (STIM) proteins arean essential component of SOC channel function, serving as the sensorsfor detecting the depletion of calcium from internal stores and foractivating SOC channels.

SUMMARY OF THE INVENTION

Described herein are compounds of Formula (I), (II), (IIA) or (IIB),compositions that include such compounds, and methods of use thereof,for modulating intracellular calcium. In one aspect, compounds ofFormula (I), (II), (IIA) or (IIB) modulate intracellular calcium byinhibition of store operated calcium channel activity. In one aspect,compounds of Formula (I), (II), (IIA) or (IIB) modulate intracellularcalcium by preventing the activity of activated store operated calciumchannel complexes. In one aspect, compounds of Formula (I), (II), (IIA)or (IIB) inhibit activation of store operated channels. In one aspect,compounds of Formula (I), (II), (IIA) or (IIB) inhibit activation ofcalcium-release activated calcium channels. In one aspect, compounds ofFormula (I), (II), (IIA) or (IIB) modulate an activity of, modulate aninteraction of, or modulate the level of, or bind to, or interact withat least one protein of the SOC channel complex. In one aspect,compounds of Formula (I), (II), (IIA) or (IIB) modulate an activity of,modulate an interaction of, or modulate the level of, or bind to, orinteract with at least one protein of the CRAC channel complex. In oneaspect, a compound of Formula (I), (II), (IIA) or (IIB) is a selectiveinhibitor of SOC channel activity. In one aspect, a compound of Formula(I), (II), (IIA) or (IIB) is a selective inhibitor of CRAC channelactivity.

In one aspect, described herein is a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof:

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R² is aryl, benzothienyl, benzofuranyl, or —CH₂CH₂-phenyl; wherein    R² is optionally substituted with 1 or 2 substituents independently    selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,    C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,    tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,    —S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸,    —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂,    —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, R¹ is hydrogen or C₁-C₆alkyl. In other embodiments, R¹ isH, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,n-pentyl, or hexyl. In yet other embodiments, R¹ is H, methyl, or ethyl.In some embodiments, R¹ is H. In one embodiment, the carboxyl moiety ofthe thiophene core is replaced with a carboxylic acid bioisostere.

In some embodiments, R² is phenyl, naphthyl, or benzothienyl; wherein R²is optionally substituted with 1 or 2 substituents independentlyselected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,C₁-C₆alkyl, C₁-C₆heteroalkyl, and C₁-C₆haloalkyl.

In some embodiments, R² is optionally substituted with 1 or 2substituents independently selected from F, Cl, Br, I, —CN, —NO₂, —OH,—CF₃, —OCF₃, —OR⁸, C₁-C₆alkyl, C₁-C₆haloalkyl, tetrazolyl,C₂-C₆heterocycloalkyl, phenyl, —N(R⁹)₂, —CO₂R⁹, —C(═O)R⁸. In someembodiments, R² is optionally substituted with 1 or 2 substituentsindependently selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃,—OMe, —OEt, -OiPr, methyl, ethyl, n-propyl, i-propyl, n-butyl,sec-butyl, t-butyl, phenyl, —NH₂, —N(Me)₂, —CO₂H, —CO₂Me, and —CO₂Et.

In some embodiments, R² is phenyl or benzothienyl; wherein R² isoptionally substituted with 1 or 2 substituents independently selectedfrom F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸, C₁-C₆alkyl,C₁-C₆heteroalkyl, and C₁-C₆haloalkyl. In other embodiments, R² is phenylor benzothienyl; wherein R² is optionally substituted with 1 or 2substituents independently selected from F, Cl, Br, I, —CN, —OH, —CF₃,—OCF₃, —OMe, methyl, ethyl, isopropyl, and t-butyl. In some embodiments,R² is selected from phenyl; 2-fluorophenyl; 3-fluorophenyl;4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl;2,4-dichlorophenyl; 2,3-dichlorophenyl; 3,4-dichlorophenyl;3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl; 4-bromophenyl;2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; and benzothien-2-yl. In otherembodiments, R² is selected from phenyl, 3-fluorophenyl; 4-fluorophenyl;4-chlorophenyl; 4-bromophenyl; 4-iodophenyl; 3-methylphenyl;3-methylphenyl; and benzothien-2-yl.

In some embodiments, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl. In other embodiments, R⁴ is a phenyl, optionallysubstituted with 1 or 2 substituents selected from F, Cl, Br, I, —CN,—CF₃, —OH, —OMe, —OCF₃, methyl, and ethyl. In yet other embodiments, R⁴is selected from phenyl; 2-fluorophenyl; 3-fluorophenyl; 4-fluorophenyl;2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl; 2,4-dichlorophenyl;2,3-dichlorophenyl; 3,4-dichlorophenyl; 3,5-dichlorophenyl;2-bromophenyl; 3-bromophenyl; 4-bromophenyl; 2-iodophenyl; 3-iodophenyl;4-iodophenyl; 2-methylphenyl; 3-methylphenyl; 4-methylphenyl;2,4-dimethylphenyl; 2,3-dimethylphenyl; 3,4-dimethylphenyl;3,5-dimethylphenyl; 2-trifluoromethylphenyl; 3-trifluoromethylphenyl;and 4-trifluoromethylphenyl. In yet some other embodiments, R⁴ isselected from phenyl; 4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl;4-chlorophenyl; 2,4-dichlorophenyl; 3,4-dichlorophenyl;3,5-dichlorophenyl; 2-bromophenyl; 4-bromophenyl; 4-methylphenyl;3,4-dimethylphenyl; and 4-trifluoromethylphenyl.

Any combination of the groups described above for the various variablesis contemplated herein.

In one aspect, the compound of Formula (I) is selected from among:

In one aspect, described herein is a method of modulating store-operatedcalcium (SOC) channel activity comprising contacting the store-operatedcalcium (SOC) channel complex, or portion thereof, with a compound ofFormula (I), (II), (IIA) or (IIB) or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof. In one aspect, the contacting occurs in vitro. Inanother aspect, the contacting occurs in vivo. In one aspect, thecompound of Formula (I), (II), (IIA) or (IIB) modulates an activity of,modulates an interaction of, or modulates the level of, or binds to, orinteracts with at least one portion of the store operated calciumchannel complex selected from stromal interaction molecules (STIM)family of proteins. In one aspect, the compound of Formula (I), (II),(IIA) or (IIB) modulates an activity of, modulates an interaction of, ormodulates the level of, or binds to, or interacts with at least oneportion of STIM1 or STIM2. In one aspect, modulating store operatedcalcium channel activity with a compound of Formula (I), (II), (IIA) or(IIB) inhibits store-operated calcium entry (SOCE). In another aspect,the store operated calcium channel complex is calcium-release activatedcalcium (CRAC) channel complex. In one aspect, modulating calciumrelease activated calcium (CRAC) activity with a compound of Formula(I), (II), (IIA) or (IIB) inhibits the electrophysiological current(I_(CRAC)) directly associated with activated CRAC channels.

In another aspect, described herein is a method of modulating calciumrelease activated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), (II), (IIA) or (IIB) orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof. In one aspect, thecompound of Formula (I), (II), (IIA) or (IIB) modulates an activity of,modulates an interaction of, or modulates the level of, or binds to, orinteracts with at least one component of the calcium release activated(CRAC) channel complex selected from stromal interaction molecules(STIM) family of proteins. In one aspect, the compound of Formula (I),(II), (IIA) or (IIB) modulates an activity of, modulates an interactionof, or modulates the level of, or binds to, or interacts with STIM1 orSTIM2. In one aspect, modulating calcium release activated calcium(CRAC) channel activity with a compound of Formula (I), (II), (IIA) or(IIB) inhibits store-operated calcium entry (SOCE). In one aspect,modulating calcium release activated calcium (CRAC) channel activitywith a compound of Formula (I), (II), (IIA) or (IIB) inhibits theelectrophysiological current (I_(CRAC)) directly associated withactivated CRAC channels. In one aspect, the compound of Formula (I),(II), (IIA) or (IIB) inhibits SOCE with an IC₅₀ below 10 μM. In yetanother aspect, the compound of Formula (I), (II), (IIA) or (IIB)inhibits electrophysiological current (I_(CRAC)) directly associatedwith activated CRAC channels at a concentration below 10 μM.

Also described herein is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), (II), (IIA) or (IIB), or pharmaceuticallyacceptable salt, pharmaceutically acceptable solvate, orpharmaceutically acceptable prodrug thereof. In one aspect, the compoundof Formula (I), (II), (IIA) or (IIB) modulates the activity of,modulates an interaction of, or binds to, or interacts with a mammalianSTIM1 protein, or a mammalian STIM2 protein. In one aspect, the disease,disorder or condition in a mammal is selected from diseases/disordersinvolving inflammation, glomerulonephritis, uveitis, hepatic diseases ordisorders, renal diseases or disorders, chronic obstructive pulmonarydisease, rheumatoid arthritis, multiple sclerosis, inflammatory boweldisease, vasculitis, dermatitis, osteoarthritis, inflammatory muscledisease, allergic rhinitis, vaginitis, interstitial cystitis,scleroderma, osteoporosis, eczema, organ transplant rejection,psoriasis, allogeneic or xenogeneic transplantation, graft rejection,graft-versus-host disease, lupus erythematosus, type I diabetes,pulmonary fibrosis, dermatomyositis, thyroiditis, myasthenia gravis,autoimmune hemolytic anemia, cystic fibrosis, chronic relapsinghepatitis, primary biliary cirrhosis, allergic conjunctivitis, hepatitisand atopic dermatitis, asthma, Sjogren's syndrome, cancer and otherproliferative diseases, and autoimmune diseases or disorders. In oneembodiment, the disease, disorder, or condition is inflammatory boweldisease. In another embodiment, the inflammatory bowel disease isulcerative colitis. In yet another embodiment, the inflammatory boweldisease is Crohn's disease. In one embodiment, the disease, disorder, orcondition is psoriasis. In one embodiment, the disease, disorder, orcondition is multiple sclerosis. In one embodiment, the disease,disorder, or condition is rheumatoid arthritis. In one embodiment, thedisease, disorder, or condition is organ transplant rejection. In yetanother aspect, the method further comprises administering to the mammala second therapeutic agent. In one aspect, the second therapeutic agentis selected from immunosuppressants, glucocorticoids, non-steroidalanti-inflammatory drugs, Cox-2-specific inhibitors, leflunomide, goldthioglucose, gold thiomalate, aurofin, sulfasalazine,hydroxychloroquinine, minocycline, anti-TNF-α agents, abatacept,anakinra, interferon-β, interferon-γ, interleukin-2, allergy vaccines,antihistamines, antileukotrienes, beta-agonists, theophylline, andanticholinergics. In another aspect, the second therapeutic agent isselected from tacrolimus, cyclosporin, rapamicin, methotrexate ,cyclophosphamide, azathioprine, mercaptopurine, mycophenolate, orFTY720, prednisone, cortisone acetate, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisone acetate, deoxycorticosterone acetate, aldosterone,aspirin, salicylic acid, gentisic acid, choline magnesium salicylate,choline salicylate, choline magnesium salicylate, choline salicylate,magnesium salicylate, sodium salicylate, diflunisal, carprofen,fenoprofen, fenoprofen calcium, fluorobiprofen, ibuprofen, ketoprofen,nabutone, ketolorac, ketorolac tromethamine, naproxen, oxaprozin,diclofenac, etodolac, indomethacin, sulindac, tolmetin, meclofenamate,meclofenamate sodium, mefenamic acid, piroxicam, meloxicam, celecoxib,rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, CS-502,JTE-522, L-745,337 and NS398, leflunomide, gold thioglucose, goldthiomalate, aurofin, sulfasalazine, hydroxychloroquinine, minocycline,infliximab, etanercept, adalimumab, abatacept, anakinra, interferon-β,interferon-γ, interleukin-2, allergy vaccines, antihistamines,antileukotrienes, beta-agonists, theophylline, and anticholinergics.

Also described herein is a method of inhibiting store-operated calciumentry (SOCE) activation of nuclear factor of activated T cells (NFAT) ina mammal comprising administering a compound of Formula (I), (II), (IIA)or (IIB), or pharmaceutically acceptable salt, pharmaceuticallyacceptable solvate, or pharmaceutically acceptable prodrug thereof. Inone aspect, the compound of Formula (I), (II), (IIA) or (IIB) modulatesan interaction of, or modulates the level of, or binds to, or interactswith a mammalian STIM1 protein, or a mammalian STIM2 protein.

Also provided herein is a method of decreasing cytokine expression byinhibiting the store-operated calcium entry activation of NFAT in amammal comprising administering a compound of Formula (I), (II), (IIA)or (IIB) or pharmaceutically acceptable salt, pharmaceuticallyacceptable solvate, or pharmaceutically acceptable prodrug thereof. Inone aspect, the compound of Formula (I), (II), (IIA) or (IIB) modulatesan interaction of, or modulates the level of, or binds to, or interactswith a mammalian STIM1 protein or a mammalian STIM2 protein. In oneaspect, the cytokine is selected from IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17,IL-18, IL-1α, IL-1β, IL-1 RA, granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),oncostatin M, erythropoietin, leukemia inhibitory factor (LIF),interferons, gamma-interferon (γ-IFN), B7.1 (CD80), B7.2 (B70, CD86),TNF-α, TNF-β, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand,4-1BBL, Trail, and migration inhibitory factor (MIF).

In one aspect, described herein is the use of a compound of Formula (I),(II), (IIA) or (IIB) or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, for the formulation of a medicament for the modulationof store operated calcium (SOC) channel activity in a subject or for thetreatment of a disease or condition in a subject that would benefit fromthe modulation of store operated calcium (SOC) channel activity. In oneaspect, the compound of Formula (I), (II), (IIA) or (IIB) inhibits storeoperated calcium entry (SOCE). In another aspect, the store operatedcalcium channel activity is calcium release activated calcium channelactivity.

Also described is an article of manufacture, comprising packagingmaterial, a compound of Formula (I), (II), (IIA) or (IIB) orcomposition, or pharmaceutically acceptable salt, pharmaceuticallyacceptable prodrug, or pharmaceutically acceptable solvate thereof,which is effective for inhibiting calcium release-activated calcium(CRAC) channel activity, or for the treatment, prevention oramelioration of one or more symptoms of a disease or condition thatwould benefit from the inhibition of calcium release-activated calcium(CRAC) channel activity, within the packaging material, and a label thatindicates that the compound or composition, or pharmaceuticallyacceptable salt, pharmaceutically acceptable prodrug, orpharmaceutically acceptable solvate thereof, is used for the inhibitionof calcium release-activated calcium (CRAC) channel activity, or for thetreatment, prevention or amelioration of one or more symptoms of adisease or condition that would benefit from the inhibition of calciumrelease-activated calcium (CRAC) channel activity. In one aspect, thecompound of Formula (I), (II), (IIA) or (IIB) inhibits store operatedcalcium entry (SOCE).

In one aspect, described herein is a compound of Formula (IIA):

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;    -   each R⁹ is independently selected from H, C₁-C₆alkyl,        C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

In one embodiment, R¹ is hydrogen or C₁-C₆alkyl. In yet another aspect,R¹ is hydrogen, methyl, ethyl, n-propyl, or iso-propyl. In oneembodiment, the carboxyl moiety of the thiophene core is replaced with acarboxylic acid bioisostere.

In another embodiment, R⁴ is a phenyl, optionally substituted with 1 or2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl.

In one embodiment, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CF₃, —OH, —OCH₃, —OCF₃,methyl, and ethyl.

In a further embodiment, the compound of Formula (IIA) is

In one aspect, described herein is a compound of Formula (IIB):

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;    -   each R⁹ is independently selected from H, C₁-C₆alkyl,        C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

In one embodiment, R¹ is hydrogen or C₁-C₆alkyl. In yet another aspect,R¹ is hydrogen, methyl, ethyl, n-propyl, or iso-propyl. In oneembodiment, the carboxyl moiety of the thiophene core is replaced with acarboxylic acid bioisostere.

In another embodiment, R⁴ is a phenyl, optionally substituted with 1 or2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl.

In one embodiment, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CF₃, —OH, —OCH₃, —OCF₃,methyl, and ethyl.

In a further aspect, the compound of Formula (IIB) is selected fromamong:

Any combination of the groups described above for the various variablesis contemplated herein.

In one aspect, described herein is a pharmaceutical compositioncomprising a pharmaceutically acceptable diluent, excipient or binder,and a compound of Formula (I), (II), (IIA) or (IIB) or pharmaceuticallyacceptable salt, pharmaceutically acceptable prodrug, orpharmaceutically acceptable solvate thereof.

In one aspect, provided herein is a pharmaceutical composition, whichincludes an effective amount of a compound provided herein, and apharmaceutically acceptable excipient. In a further aspect, provided arecompositions further including a second pharmaceutically activeingredient.

In certain embodiments, provided herein is a pharmaceutical compositioncontaining: i) a physiologically acceptable carrier, diluent, and/orexcipient; and ii) one or more compounds described herein.

In any of the aforementioned aspects are further embodiments thatinclude single administrations of the effective amount of the compoundof Formula (I), (II), (IIA) or (IIB), including further embodiments inwhich: (i) the compound of Formula (I), (II), (IIA) or (IIB) isadministered once; (ii) the compound of Formula (I), (II), (IIA) or(IIB) is administered to the mammal multiple times over the span of oneday; (iii) continually; or (iv) continuously.

In any of the aforementioned aspects are further embodiments thatinclude multiple administrations of the effective amount of the compoundof Formula (I), (II), (IIA) or (IIB), including further embodiments inwhich (i) the compound of Formula (I), (II), (IIA) or (IIB) isadministered in a single dose; (ii) the time between multipleadministrations is every 6 hours; (iii) the compound of Formula (I),(II), (IIA) or (IIB) is administered to the mammal every 8 hours. Infurther or alternative embodiments, the method comprises a drug holiday,wherein the administration of the compound of Formula (I), (II), (IIA)or (IIB) is temporarily suspended or the dose of the compound of Formula(I), (II), (IIA) or (IIB) being administered is temporarily reduced; atthe end of the drug holiday, dosing of the compound of Formula (I),(II), (IIA) or (IIB) is resumed. The length of the drug holiday can varyfrom 2 days to 1 year.

In one aspect, compounds of Formula (I), (II), (IIA) or (IIB) describedherein are administered to a human. In some embodiments, compounds ofFormula (I), (II), (IIA) or (IIB) described herein are orallyadministered.

Compounds provided herein are used for modulating intracellular calcium.In one aspect, compounds provided herein modulate SOC channel activity.In one aspect, compounds provided herein modulate CRAC channel activity.In another aspect, compounds provided herein modulate STIM proteinactivity. In another aspect, compounds provided herein modulate Oraiprotein activity. In another aspect, compounds provided herein modulatethe functional interactions of STIM proteins with Orai proteins. Inanother aspect, compounds provided herein reduce the number offunctional SOC channels. In another aspect, compounds provided hereinreduce the number of functional CRAC channels. In one aspect, compoundsdescribed herein are SOC channel blockers. In one aspect, compoundsdescribed herein are CRAC channel blockers.

In one aspect, compounds of Formula (I), (II), (IIA) or (IIB) areselective inhibitors of SOCE. In one aspect, compounds of Formula (I),(II), (IIA) or (IIB) are selective inhibitors of CRAC channel activity.

Other objects, features and advantages of the compounds, compositions,methods, and uses described herein will become apparent from thefollowing detailed description. It should be understood, however, thatthe detailed description and the specific examples, while indicatingspecific embodiments, are given by way of illustration only, sincevarious changes and modifications within the spirit and scope of thedisclosure will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 outlines the I_(CRAC) channel pathway.

FIG. 2 shows that Compound 1(2-(4-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid)produces a concentration-dependent inhibition of the initial kinetics ofthe CRAC channel response in RBL-2H3 cells.

FIG. 3 shows the results of treatment of RBL-2H3 cells with Compound 1.Activation of CRAC channels and downstream effectors was induced inRBL-2H3 cells with TG/TPA and the effects of Compound 1 were examined ondegranulation and TNF-alpha release. Compound 1 inhibited degranulationwith an IC₅₀ of 2.6 μM and inhibited TNF-alpha release with an IC₅₀ of3.3 μM.

FIG. 4 shows the results of a degranulation assay in RBL-2H3 cells. A 2hour TG/TPA induction in Hanks-buffered salt solution (HBSS) or HBSS+1%serum was preceded by a 10 minute pretreatment with Compound 1. In theabsence of serum, Compound 1 inhibited degranulation with an apparentIC₅₀ less than about 0.3 μM, whereas in the presence of serum, Compound1 inhibited degranulation with an IC₅₀ of about 1-3 μM.

FIG. 5 shows the effects of Compound 1 on SOCE, cell proliferation, andIL-2 secretion in rat T cells. Compound 1 inhibited SOCE with an IC₅₀ ofabout 2.4 μM, inhibited cell proliferation with an IC₅₀ of about 4.3 μM,and inhibited IL-2 secretion with an IC₅₀ of about 2.5 μM.

FIG. 6 shows the results of inhibition of Con A-induced cellproliferation of primary human T cells with Compound 1. Compound 1inhibited Con A-induced cell proliferation of primary human T cells withan IC₅₀ of about 3.7 μM.

FIG. 7 shows the results of inhibition of IL-2 release in Jurkat T cellswith Compound 1. Jurkat T cells were stimulated with PHA+TPA in thepresence of Compound 1. Compound 1 inhibited IL-2 release from Jurkat Tcells with an IC₅₀ of about 0.5 μM.

FIG. 8 shows the selectivity profile of Compound 1. Compound 1 is aselective inhibitor of SOCE.

FIG. 9 shows the selectivity of Compound 1 for SOC channels vs. voltagegated calcium channels expressed in human SH-SY5Y cells.

FIG. 10 shows the additive effects of Compound 1 and cyclosporin A ininhibiting IL-2 secretion from Jurkat T cells. Jurkat T cells werestimulated with PHA/TPA in order to induce IL-2 secretion.

FIG. 11 shows the results of orally administered Compound 1 in the ratmodel of collagen induced arthritis. In the same assay, cyclosporin Agiven orally produced a 9% reduction in liver weight, whereas no liverweight reduction was seen in the rats treated with Compound 1.

FIG. 12 shows the results of orally administered Compound 1 in the mousedelayed-type hypersensitivity (DTH) model.

FIG. 13 shows results of treatment of HEK293 overexpressing STIM1 withCompound 1 and 2-APB. Overexpression of STIM1 in HEK293 cells enhances aCa²⁺ entry pathway that resembles the endogenous SOCE (see theExamples). The STIM1-dependent Ca²⁺ entry signal is blocked by Compound1 in a concentration dependent manner and 2-APB both enhances (at lowconcentrations) and inhibits (at high concentrations) the signal.

FIG. 14 shows the results of inhibition of recombinanthOrail/hSTIM1-dependent Ca²⁺ entry in stable cells with Compound 1 andCompound 2.

FIG. 15 shows that Compound 1 and Compound 2 inhibit the MonsterI_(CRAC) expressed in cells stably overexpressing hOrail/hSTIM1. Forcomparison, the effects of some reference compounds are also shown.

FIG. 16 shows Compound 1 inhibits recombinant Ca_(v)1.2 L-type Ca²⁺channels.

FIG. 17 shows the oral bioavailability and T_(1/2) of Compound 1 inrats.

FIG. 18 shows the results of delayed rejection of skin transplants inLewis rats treated with Compound 1.

FIG. 19 shows the results of 24-hour plasma exposures of Compound 1after oral administration at 10 or 50 mg/kg. Each point represents themean of three samples unless noted otherwise.

FIG. 20 shows the results of inhibition of inflammation in the ratulcerative colitis model for IBD with Compound 1.

FIG. 21 shows the bioavailability and T_(1/2) of Compound 2(2-(3-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid) inrats, and the effect of orally administered Compound 2 in the mouse DTHmodel of inflammation.

DETAILED DESCRIPTION

Cellular calcium homeostasis is a result of the summation of regulatorysystems involved in the control of intracellular calcium levels andmovements. Cellular calcium homeostasis is achieved, at least in part,by calcium binding and by movement of calcium into and out of the cellacross the plasma membrane and within the cell by movement of calciumacross membranes of intracellular organelles including, for example, theendoplasmic reticulum, sarcoplasmic reticulum, mitochondria andendocytic organelles including endosomes and lysosomes.

Movement of calcium across cellular membranes is carried out byspecialized proteins. For example, calcium from the extracellular spacecan enter the cell through various calcium channels and a sodium/calciumexchanger and is actively extruded from the cell by calcium pumps andsodium/calcium exchangers. Calcium can also be released from internalstores through inositol trisphosphate or ryanodine receptors and can betaken up by these organelles by means of calcium pumps.

Calcium can enter cells by any of several general classes of channels,including but not limited to, voltage-operated calcium (VOC) channels,store-operated calcium (SOC) channels, and sodium/calcium exchangersoperating in reverse mode. VOC channels are activated by membranedepolarization and are found in excitable cells like nerve and muscleand are for the most part not found in nonexcitable cells. Under someconditions, Ca²⁺ can enter cells via Na⁺—Ca²⁺ exchangers operating inreverse mode.

Endocytosis provides another process by which cells can take up calciumfrom the extracellular medium through endosomes. In addition, somecells, e.g., exocrine cells, can release calcium via exocytosis.

Cytosolic calcium concentration is tightly regulated with resting levelsusually estimated at approximately 0.1 μM in mammalian cells, whereasthe extracellular calcium concentration is typically about 2 mM. Thistight regulation facilitates transduction of signals into and withincells through transient calcium flux across the plasma membrane andmembranes of intracellular organelles. There is a multiplicity ofintracellular calcium transport and buffer systems in cells that serveto shape intracellular calcium signals and maintain the low restingcytoplasmic calcium concentration. In cells at rest, the principalcomponents involved in maintaining basal calcium levels are calciumpumps and leaks in the endoplasmic reticulum and plasma membrane.Disturbance of resting cytosolic calcium levels can effect transmissionof such signals and give rise to defects in a number of cellularprocesses. For example, cell proliferation involves a prolonged calciumsignaling sequence. Other cellular processes include, but are notlimited to, secretion, signalling, and fertilization, involve calciumsignaling.

Cell-surface receptors that activate phospholipase C (PLC) createcytosolic Ca²⁺ signals from intra- and extra-cellular sources. Aninitial transient rise of [Ca²⁺]_(i) (intracellular calciumconcentration) results from the release of Ca²⁺ from the endoplasmicreticulum (ER), which is triggered by the PLC product,inositol-1,4,5-trisphosphate (IP₃), opening IP₃ receptors in the ER(Streb et al. Nature, 306, 67-69, 1983). A subsequent phase of sustainedCa²⁺ entry across the plasma membrane then ensues, through specializedstore operated calcium (SOC) channels (in the case of immune cells theSOC channels are calcium release-activated calcium (CRAC) channels) inthe plasma membrane. Store-operated Ca²⁻ entry (SOCE) is the process inwhich the emptying of Ca²⁺ stores itself activates Ca²⁺ channels in theplasma membrane to help refill the stores (Putney, Cell Calcium, 7,1-12, 1986; Parekh et al., Physiol. Rev. 757-810; 2005). SOCE does morethan simply provide Ca²⁺ for refilling stores, but can itself generatesustained Ca²⁺ signals that control such essential functions as geneexpression, cell metabolism and exocytosis (Parekh and Putney, Physiol.Rev. 85, 757-810 (2005).

In lymphocytes and mast cells, activation of antigen or Fc receptorscauses the release of Ca²⁺ from intracellular stores, which in turnleads to Ca²⁺ influx through CRAC channels in the plasma membrane. Thesubsequent rise in intracellular Ca²⁺ activates calcineurin, aphosphatase that regulates the transcription factor NFAT. In restingcells, NFAT is phosphorylated and resides in the cytoplasm, but whendephosphorylated by calcineurin, NFAT translocates to the nucleus andactivates different genetic programmes depending on stimulationconditions and cell type. In response to infections and duringtransplant rejection, NFAT partners with the transcription factor AP-1(Fos-Jun) in the nucleus of “effector” T cells, thereby transactivatingcytokine genes, genes that regulate T cell proliferation and other genesthat orchestrate an active immune response (Rao et al., Annu RevImmunol., 1997; 15:707-47). In contrast, in T cells recognizing selfantigens, NFAT is activated in the absence of AP-1, and activates atranscriptional programme known as “anergy” that suppresses autoimmuneresponses (Macian et al., Transcriptional mechanisms underlyinglymphocyte tolerance. Cell. 2002 Jun 14; 109(6):719-31). In a subclassof T cells known as regulatory T cells which suppress autoimmunitymediated by self-reactive effector T cells, NFAT partners with thetranscription factor FOXP3 to activate genes responsible for suppressorfunction (Wu et al., Cell, 2006 Jul. 28; 126(2):375-87; Rudensky A Y,Gavin M, Zheng Y. Cell. 2006 Jul 28;126(2):253-256).

The endoplasmic reticulum (ER) carries out a variety processes. The ERhas a role as both an agonist-sensitive Ca²⁺ store and sink, proteinfolding/processing takes place within its lumen. Here, numerousCa²⁺-dependent chaperone proteins ensure that newly synthesized proteinsare folded correctly and sent off to the appropriate destination. The ERis also involved in vesicle trafficking, release of stress signals,regulation of cholesterol metabolism, and apoptosis. Many of theseprocesses require intraluminal Ca²⁺, and protein misfolding, ER stressresponses, and apoptosis can all be induced by depleting the ER of Ca²⁺for prolonged periods of time. Because of its role as a source of Ca²⁺,it is clear that ER Ca²⁺ content must fall after stimulation. However,to preserve the functional integrity of the ER, it is vital that theCa²⁺ content does not fall too low or is maintained at a low level.Replenishment of the ER with Ca²⁺ is therefore a central process to alleukaryotic cells. Because a fall in ER Ca²⁺ content activatesstore-operated Ca²⁺ channels in the plasma membrane, a major function ofthis Ca²⁺ entry pathway is believed to be maintenance of ER Ca²⁺ levelsthat are necessary for proper protein synthesis and folding. However,store-operated Ca²⁺ channels have other important roles.

The understanding of store operated calcium entry was provided byelectrophysiological studies which established that the process ofemptying the stores activated a Ca²⁺ current in mast cells called Ca²⁺release-activated Ca²⁺ current or I_(CRAC). I_(CRAC) is non-voltageactivated, inwardly rectifying, and remarkably selective for Ca²⁺. It isfound in several cell types mainly of hemapoietic origin. I_(CRAC) isnot the only store-operated current, and it is now apparent thatstore-operated influx encompasses a family of Ca²⁺-permeable channels,with different properties in different cell types. I_(CRAC) was thefirst store-operated Ca²⁺ current to be described and remains a popularmodel for studying store-operated influx.

Store-operated calcium channels can be activated by any procedure thatempties the stores; it does not seem to matter how the stores areemptied, the net effect is activation of store-operated Ca²⁺ entry.Physiologically, store emptying is evoked by an increase in the levelsof IP₃ or other Ca²⁺-releasing signals followed by Ca²⁺ release from thestores. But there are several other methods for emptying stores. Thesemethods include the following:

-   1) elevation of IP₃ in the cytosol (following receptor stimulation    or, dialyzing the cytosol with IP₃ itself or related congeners like    the nonmetabolizable analog Ins(2,4,5)P₃);-   2) application of the Ca²⁺ ionophore ionomycin to permeabilize the    ER membrane;-   3) dialyzing the cytoplasm with high concentrations of the Ca²⁺    chelators EGTA or BAPTA, which chelate Ca² that leaks from the    stores and hence prevent store refilling;-   4) exposure to the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase    (SERCA) inhibitors like thapsigargin, cyclopiazonic acid, and    di-tert-butylhydroquinone which prevent the P-type ATPases from    refilling the stores;-   5) sensitizing the IP₃ receptors to resting levels of InsP₃ with    agents like thimerosal; and-   6) loading membrane-permeable metal Ca²⁻ chelators like    N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN) directly    into the stores.

Through mass action, TPEN lowers free intraluminal Ca2+ concentrationwithout changing total store Ca²⁺ such that the storedepletion-dependent signal is generated.

These methods of emptying stores are not devoid of potential problems.The key feature of store-operated Ca²⁺ entry is that it is the fall inCa²⁻ content within the stores and not the subsequent rise incytoplasmic Ca²⁺ concentration that activates the channels. However,ionomycin and SERCA pump blockers generally cause a rise in cytoplasmicCa²⁺ concentration as a consequence of store depletion, and such a risein Ca²⁺ could open Ca⁻-activated cation channels permeable to Ca⁺. Oneway to avoid such problems is to use agents under conditions wherecytoplasmic Ca²⁺ has been strongly buffered with high concentrations ofCa²⁺ chelator such as EGTA or BAPTA.

Store-Operated Calcium Entry

Reduced calcium concentration in intracellular calcium stores such asthe endoplasmic reticulum resulting from release of calcium therefromprovides a signal for influx of calcium from the extracellular mediuminto the cell. This influx of calcium, which produces a sustained“plateau” elevation of cytosolic calcium concentration, generally doesnot rely on voltage-gated plasma membrane channels and does not involveactivation of calcium channels by calcium. This calcium influx mechanismis referred to as capacitative calcium entry (CCE), calciumrelease-activated, store-operated or depletion-operated calcium entry.Store-operated calcium entry can be recorded as an ionic current withdistinctive properties. This current is referred to as I_(soc)(store-operated current) or I_(CRAC) (calcium release-activatedcurrent).

Electrophysiological analysis of store-operated or calciumrelease-activated currents reveals distinct biophysical properties (see,e.g., Parekh and Penner (1997) Physiol. Rev. 77:901-930) of thesecurrents. For example, the current can be activated by depletion ofintracellular calcium stores (e.g., by nonphysiological activators suchas thapsigargin, CPA, ionomycin and BAPTA, and physiological activatorssuch as IP₃) and can be selective for divalent cations, such as calcium,over monovalent ions in physiological solutions or conditions, can beinfluenced by changes in cytosolic calcium levels, and can show alteredselectivity and conductivity in the presence of low extracellularconcentrations of divalent cations. The current may also be blocked orenhanced by 2-APB (depending on concentration) and blocked by SKF96365and Gd³⁺ and generally can be described as a calcium current that is notstrictly voltage-gated.

Patch-clamp studies in mast cells and Jurkat leukaemic T cells haveestablished the CRAC entry mechanism as an ion channel with distinctivebiophysical characteristics, including a high selectivity for Ca²⁺paired with an exceedingly low conductance. Furthermore, the CRACchannel was shown to fulfill the rigorous criteria for beingstore-operated, which is the activation solely by the reduction of Ca²⁺in the ER rather than by cytosolic Ca²⁺ or other messengers generated byPLC (Prakriya et al., In Molecular and Cellular Insights into IonChannel Biology (ed. Robert Maue) 121-140 (Elsevier Science, Amsterdam,2004)).

Regulation of Store-Operated Calcium Entry by Intracellular CalciumStores

Store-operated calcium entry is regulated by the level of calcium withinan intracellular calcium store. Intracellular calcium stores can becharacterized by sensitivity to agents, which can be physiological orpharmacological, which activate release of calcium from the stores orinhibit uptake of calcium into the stores. Different cells have beenstudied in characterization of intracellular calcium stores, and storeshave been characterized as sensitive to various agents, including, butnot limited to, IP₃ and compounds that effect the IP₃ receptor,thapsigargin, ionomycin and/or cyclic ADP-ribose (cADPR) (see, e.g.,Berridge (1993) Nature 361:315-325; Churchill and Louis (1999) Am. J.Physiol. 276:C426-C434; Dargie et al. (1990) Cell Regul. 1:279-290;Gerasimenko et al. (1996) Cell 84:473-480; Gromoda et al. (1995) FEBSLett. 360:303-306; Guse et al. (1999) Nature 398:70-73).

Accumulation of calcium within endoplasmic reticulum and sarcoplasmicreticulum (SR; a specialized version of the endoplasmic reticulum instriated muscle) storage organelles is achieved throughsarcoplasmic-endoplasmic reticulum calcium ATPases (SERCAs), commonlyreferred to as calcium pumps. During signaling (i.e., when endoplasmicreticulum channels are activated to provide for calcium release from theendoplasmic reticulum into the cytoplasm), endoplasmic reticulum calciumis replenished by the SERCA pump with cytoplasmic calcium that hasentered the cell from the extracellular medium (Yu and Hinkle (2000) J.Biol. Chem. 275:23648-23653; Hofer et al. (1998) EMBO J. 17:1986-1995).

Calcium release channels associated with IP₃ and ryanodine receptorsprovide for controlled release of calcium from endoplasmic andsarcoplasmic reticulum into the cytoplasm resulting in transientincreases in cytoplasmic calcium concentration. IP₃ receptor-mediatedcalcium release is triggered by IP₃ formed in the break down of plasmamembrane phosphoinositides through the action of phospholipase Cactivated by binding of an agonist to a plasma membrane Gprotein-coupled receptor. Ryanodine receptor-mediated calcium release istriggered by an increase in cytoplasmic calcium and is referred to ascalcium-induced calcium release (CICR). The activity of ryanodinereceptors (which have affinity for ryanodine and caffeine) may also beregulated by cyclic ADP-ribose.

Thus, the calcium levels in the stores, and in the cytoplasm, fluctuate.For example, ER free calcium can decrease from a range of about 60-400μM to about 1-50 μM when HeLa cells are treated with histamine, anagonist of PLC-linked histamine receptors (Miyawaki et al. (1997) Nature388:882-887). Store-operated calcium entry is activated as the freecalcium concentration of the intracellular stores is reduced. Depletionof store calcium, as well as a concomitant increase in cytosolic calciumconcentration, can thus regulate store-operated calcium entry intocells.

Cytoplasmic Calcium Buffering

Agonist activation of signaling processes in cells can involve dramaticincreases in the calcium permeability of the endoplasmic reticulum, forexample, through opening of IP₃ receptor channels, and the plasmamembrane through store-operated calcium entry. These increases incalcium permeability are associated with an increase in cytosoliccalcium concentration that can be separated into two components: a“spike” of calcium release from the endoplasmic reticulum duringactivation of the IP₃ receptor and a plateau phase which is a sustainedelevation of calcium levels resulting from entry of calcium into thecytoplasm from the extracellular medium. Upon stimulation, the restingintracellular free calcium concentration of about 100 nM can riseglobally to greater than 1 μM. The cell modulates these calcium signalswith endogenous calcium buffers, including physiological buffering byorganelles such as mitochondria, endoplasmic reticulum and Golgi.Mitochondrial uptake of calcium through a uniporter in the innermembrane is driven by the large negative mitochondrial membranepotential, and the accumulated calcium is released slowly throughsodium-dependent and -independent exchangers, and, under somecircumstances, the permeability transition pore (PTP). Thus,mitochondria can act as calcium buffers by taking up calcium duringperiods of activation and slowly releasing it later. Uptake of calciuminto the endoplasmic reticulum is regulated by the sarcoplasmic andendoplasmic reticulum calcium ATPase (SERCA). Uptake of calcium into theGolgi is mediated by a P-type calcium transport ATPase (PMR1/ATP2C1).Additionally, there is evidence that a significant amount of the calciumreleased upon IP₃ receptor activation is extruded from the cell throughthe action of the plasma membrane calcium ATPase. For example, plasmamembrane calcium ATPases provide the dominant mechanism for calciumclearance in human T cells and Jurkat cells, although sodium/calciumexchange also contributes to calcium clearance in human T cells. Withincalcium-storing organelles, calcium ions can be bound to specializedcalcium-buffering proteins, such as, for example, calsequestrins,calreticulins and calnexins. Additionally, there are calcium-bufferingproteins in the cytosol that modulate calcium spikes and assist inredistribution of calcium ions. Thus, proteins and other molecules thatparticipate in any of these and other mechanisms through which cytosoliccalcium levels can be reduced are proteins that are involved in,participate in and/or provide for cytoplasmic calcium buffering. Thus,cytoplasmic calcium buffering allows for sustained calcium influxthrough SOC channels. Large increases in cytoplasmic Ca2+ or storerefilling deactivate SOCE.

Downstream Calcium Entry-Mediated Events

In addition to intracellular changes in calcium stores, store-operatedcalcium entry affects a multitude of events that are consequent to or inaddition to the store-operated changes. For example Ca²⁺ influx resultsin the activation of a large number of calmodulin-dependent enzymesincluding the serine phosphatase calcineurin. Activation of calcineurinby an increase in intracellular calcium results in acute secretoryprocesses such as mast cell degranulation. Activated mast cells releasepreformed granules containing histamine, heparin, TNFα and enzymes suchas β-hexosaminidase. Some cellular events, such as B and T cellproliferation, require sustained calcineurin signaling, which requires asustained increase in intracellular calcium. A number of transcriptionfactors are regulated by calcineurin, including NFAT (nuclear factor ofactivated T cells), MEF2 and NFKB. NFAT transcription factors playimportant roles in many cell types, including immune cells. In immunecells NFAT mediates transcription of a large number of molecules,including cytokines, chemokines and cell surface receptors.Transcriptional elements for NFAT have been found within the promotersof cytokines such as IL-2, IL-3, IL-4, IL-5, IL-8, IL-13, as well astumor necrosis factor alpha (TNFα), granulocyte colony-stimulatingfactor (G-CSF), and gamma-interferon (γ-IFN).

The activity of NFAT proteins is regulated by their phosphorylationlevel, which in turn is regulated by both calcineurin and NFAT kinases.Activation of calcineurin by an increase in intracellular calcium levelsresults in dephosphorylation of NFAT and entry into the nucleus.Rephosphorylation of NFAT masks the nuclear localization sequence ofNFAT and prevents its entry into the nucleus. Because of its strongdependence on calcineurin-mediated dephosphorylation for localizationand activity, NFAT is a sensitive indicator of intracellular calciumlevels.

Diseases, Disorders or Conditions

Clinical studies demonstrate that the CRAC channel is absolutelyrequired for the activation of genes underlying the T cell response toantigen. Sustained calcium entry is needed for lymphocyte activation andadaptive immune response. Calcium entry into lymphocytes occursprimarily through the CRAC channels. Increased calcium leads to NFATactivation and expression of cytokines required for immune response.Inhibiting the store operated calcium entry is an efficient way toprevent T cell activation.

Inhibition of CRAC channel activity with the compounds described herein,such as compounds of Formula (I), (IIA), and (IIB) provide a means forproviding immunosuppresive therapy as demonstrated by the elimination ofstore-operated calcium entry noted in patients with severe-combinedimmunodeficiency (SCID). T cells, fibroblasts, and in some cases Bcells, from patients with T cell immunodeficiency or SCID having aprincipal defect in T cell activation show a strong defect instore-operated calcium entry (Feske et al. (2001) Nature Immunol.2:316-324; Paratiseti et al. (1994) J. Biol. Chem. 269:32327-32335; andLe Deist et al. (1995) Blood 85:1053-1062). SCID patients lack adaptiveimmune response, but without any impairment or toxicity in major organs.The SCID patient phenotype indicates that inhibition of CRAC channels isan effective strategy for immunosuppression.

Diseases/Disorders Involving Inflammation and Diseases/Disorders Relatedto the Immune System

Diseases or disorders that can be treated or prevented using thecompounds, compositions, and methods provided herein include diseasesand disorders involving inflammation and/or that are related to theimmune system. These diseases include but are not limited to asthma,chronic obstructive pulmonary disease, rheumatoid arthritis,inflammatory bowel disease, glomerulonephritis, neuroinflammatorydiseases such as multiple sclerosis, and disorders of the immune system.

The activation of neutrophils (PMN) by inflammatory mediators is partlyachieved by increasing cytosolic calcium concentration. Store-operatedcalcium influx in particular is thought to play an important role in PMNactivation. It has been shown that trauma increases PMN store-operatedcalcium influx (Hauser et al. (2000) J. Trauma Injury Infection andCritical Care 48 (4):592-598) and that prolonged elevations of cytosoliccalcium concentration due to enhanced store-operated calcium influx mayalter stimulus-response coupling to chemotaxins and contribute to PMNdysfunction after injury. Modulation of PMN cytosolic calciumconcentration through store-operated calcium channels might therefore beuseful in regulating PMN-mediated inflammation and spare cardiovascularfunction after injury, shock or sepsis (Hauser et al. (2001) J.Leukocyte Biology 69 (1):63-68).

Calcium plays a critical role in lymphocyte activation. Activation oflymphocytes, e.g., by antigen stimulation, results in rapid increases inintracellular free calcium concentrations and activation oftranscription factors, including nuclear factor of activated T cells(NFAT), NF-κB, JNK1, MEF2 and CREB. NFAT is a key transcriptionalregulator of the IL-2 (and other cytokine) genes (see, e.g. Lewis (2001)Annu. Rev. Immunol 19:497-521). A sustained elevation of intracellularcalcium level is required to keep NFAT in a transcriptionally activestate, and is dependent on store-operated calcium entry. Reduction orblocking of store-operated calcium entry in lymphocytes blockscalcium-dependent lymphocyte activation. Thus, modulation ofintracellular calcium, and particularly store-operated calcium entry(e.g., reduction in, elimination of store-operated calcium entry), inlymphocytes can be a method for treating immune and immune-relateddisorders, including, for example, chronic immune diseases/disorders,acute immune diseases/disorders, autoimmune and immunodeficiencydiseases/disorders, diseases/disorders involving inflammation, organtransplant graft rejections and graft-versus-host disease and altered(e.g., hyperactive) immune responses. For example treatment of anautoimmune disease/disorder might involve reducing, blocking oreliminating store-operated calcium entry in lymphocytes.

Examples of immune disorders include psoriasis, rheumatoid arthritis,vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis,asthma, inflammatory muscle disease, allergic rhinitis, vaginitis,interstitial cystitis, scleroderma, osteoporosis, eczema, allogeneic orxenogeneic transplantation (organ, bone marrow, stem cells and othercells and tissues) graft rejection, graft-versus-host disease, lupuserythematosus, inflammatory disease, type I diabetes, pulmonaryfibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g.,Hashimoto's and autoimmune thyroiditis), myasthenia gravis, autoimmunehemolytic anemia, multiple sclerosis, cystic fibrosis, chronic relapsinghepatitis, primary biliary cirrhosis, allergic conjunctivitis and atopicdermatitis.

Cancer and Other Proliferative Diseases

Compounds of Formula (I), (II), (II), (IIA) and (IIB), compositionsthereof, and methods provided herein may be used in connection withtreatment of malignancies, including, but not limited to, malignanciesof lymphoreticular origin, bladder cancer, breast cancer, colon cancer,endometrial cancer, head and neck cancer, lung cancer, melanoma, ovariancancer, prostate cancer and rectal cancer. Store-operated calcium entrymay play an important role in cell proliferation in cancer cells (Weisset al. (2001) International Journal of Cancer 92 (6):877-882).

Inhibition of SOCE is sufficient to prevent tumor cell proliferation.The pyrazole derivative BTP-2, a direct I_(CRAC) blocker inhibits SOCEand proliferation in Jurkat cells (Zitt et al., J. Biol. Chem., 279,12427-12437, 2004) and in colon cancer cells. It has been suggested thatsustained SOCE requires mitochonrial Ca²⁺ uptake (Nunez et al., J.Physiol. 571.1, 57-73, 2006) and that prevention of mitochondrial Ca²⁺uptake leads to SOCE inhibition (Hoth et al., P.N.A.S., 97, 10607-10612,2000; Hoth et al., J. Cell. Biol. 137, 633-648, 1997; Glitsch et al.,EMBO J., 21, 6744-6754, 2002). Stimulation of Jurkat cells inducessustained SOCE and activation of the Ca²⁺-dependent phosphatasecalcineurin that dephosphorylates NFAT, promoting expression ofinterleukin-2 and proliferation. Compounds of Formula (I), (II), (IIA)and (IIB) inhibit SOCE and may be used in the treatment of cancer orother proliferative diseases or conditions.

Liver Diseases and Disorders

Diseases or disorders that can be treated or prevented using thecompounds of Formula (I), (II), (IIA) or (IIB), compositions thereof,and methods provided herein include hepatic or liver diseases anddisorders. These diseases and disorders include but are not limited toliver injury, for example, due to transplantation, hepatitis andcirrhosis.

Store-operated calcium entry has been implicated in chronic liverdisease (Tao et al. (1999) J. Biol. Chem., 274(34):23761-23769) as wellas transplantation injury after cold preservation-warm reoxygenation(Elimadi et al. (2001) Am J. Physiology, 281(3 Part 1):G809-G815).

Kidney Diseases and Disorders

Diseases or disorders that can be treated or prevented using the methodsprovided herein include kidney or renal diseases and disorders.Mesangial cell hyperplasia is often a key feature of such diseases anddisorders. Such diseases and disorders may be caused by immunological orother mechanisms of injury, including IgAN, membranoproliferativeglomerulonephritis or lupus nephritis. Imbalances in the control ofmesangial cell replication also appear to play a key role in thepathogenesis of progressive renal failure.

The turnover of mesangial cells in normal adult kidney is very low witha renewal rate of less than 1%. A prominent feature of glomerular/kidneydiseases is mesangial hyperplasia due to elevated proliferation rate orreduced cell loss of mesangial cells. When mesangial cell proliferationis induced without cell loss, for example due to mitogenic stimulation,mesangioproliferative glomerulonephritis can result. Data have indicatedthat regulators of mesangial cell growth, particularly growth factors,may act by regulating store-operated calcium channels (Ma et al. (2001)J Am. Soc. of Nephrology, 12:(1) 47-53). Modulators of store-operatedcalcium influx may aid in the treatment of glomerular diseases byinhibiting mesangial cell proliferation.

Store Operated Calcium Channels

Clinical studies demonstrate that the CRAC channel, a type of SOCchannel, is absolutely required for the activation of genes underlyingthe T cell response to antigen (Partiseti et al., J Biol. Chem., 269,32327-32335, 1994; Feske et al., Curr. Biol. 15, 1235-1241, 2005). SOCEcan contribute directly to the elevation of cytosolic Ca²⁺ levels([Ca²⁺]_(i)), as in T lymphocytes where CRAC channels generate thesustained Ca²⁺ signals needed to drive gene expression underlying T cellactivation by antigen. Sustained calcium entry is needed for lymphocyteactivation and adaptive immune response. Calcium entry into lymphocytesoccurs primarily through the CRAC channels. Increased calcium levelslead to NFAT activation and expression of cytokines required for immuneresponse.

The CRAC channel has a distinctive biophysical fingerprint, quantifiablestore-dependence, and essential function in T cells. Studies have shownthat CRAC channels are formed from two component proteins, whichinteract to form CRAC channels. The CRAC channel is assembled by twofunctional components, STIM1 and Orail. STIM1 (stromal interactionmolecule 1) was identified as the mammalian ER Ca²⁺ sensor (Liou, J. etal. Curr. Biol. 15, 1235-1241 (2005); Roos, J. et al. J. Cell Biol. 169,435-445 (2005); WO 20041078995; US 2007/0031814). Orai1/CRACM1 wasidentified as a component of the mammalian CRAC channel (Feske, S. etal. Nature 441, 179-185 (2006); Vig, M. et al. Science 312, 1220-1223(2006); Zhang, S. L. et al. Proc. Natl Acad. Sci. USA 103, 9357-9362(2006)).

STIM1 is the sensor of Ca²⁺ within ER Ca²⁺ stores, moving in response tostore depletion into ER puncta close to the plasma membrane. Orail is apore forming CRAC channel subunit in the plasma membrane. The twomembrane proteins STIM1 and Orail have each been shown to be essentialfor the activation of CRAC channels.

Expression of both STIM1 and Orail in human embryonic kidney 293 cells(HEK293 cells) reconstitute functional CRAC channels. Expression ofOrail alone strongly reduces store-operated Ca²⁺ entry in HEK293 cellsand the Ca²⁺ release-activated Ca²⁺ current (I_(CRAC)) in rat basophilicleukemia cells. However, expressed along with the store-sensing STIM1protein, Orai1 causes a massive increase in SOCE, enhancing the rate ofCa²⁺ entry by up to 103-fold. This entry is entirely store dependentsince the same coexpression causes no measurable store-independent Ca²⁺entry. The entry is completely blocked by the store operated channelblocker, 2-aminoethoxydiphenylborate. STIM proteins are known to mediateCa²⁺ store-sensing and endoplasmic reticulum-plasma membrane couplingwith no intrinsic channel properties. Orail contributes the plasmamembrane channel component responsible for Ca²⁺ entry. The suppressionof CRAC channel function by Orai1 overexpression reflects a requiredstoichiometry between STIM1 and Orai1 (Soboloff et al., J. Biol. Chem.Vol. 281, no. 30, 20661-20665, 2006).

Stromal Interacting Molecule (STIM) Proteins

In RNAi screen in Drosophila S2 cells using thapsigargin-activated Ca²⁺entry as a marker for store-operated channels, one gene gave asubstantially reduced Ca²⁺ entry, coding for the protein stromalinteraction molecule (Stim) (Roos, J. et al. J. Cell Biol. 169, 435-445,2005). There are two homologues of Stim in mammalian cells, STIM1 andSTIM2, both of which appear to be distributed ubiquitously (Williams etal., Biochem J. 2001 Aug. 1; 357(Pt 3):673-85). STIM1 is the ER Ca²⁺sensor for store-operated Ca²⁺ entry. STIM1 is a 77 kDa type I membraneprotein with multiple predicted protein interaction or signallingdomains and is located predominantly in the ER, but also to a limitedextent in the plasma membrane.

Knockdown of STIM1 by RNAi substantially reduced I_(CRAC) in Jurkat Tcells, and store-operated Ca²⁺ entry in HEK293 epithelial cells andSH—SY5Y neuroblastoma cells. However, knockdown of the closely relatedSTIM2 had no effect. These results indicate an essential role of STIM(Drosophila) and STIM1 (mammals) in the mechanism of activation ofstore-operated channels. It is unlikely that STIM1 is the store-operatedchannel itself. It has no channel-like sequence, and overexpression ofthe protein only modestly enhances Ca²⁺ entry. STIM1 is located both onthe plasma and intracellular membranes, such as the ER (Manji et al.,Biochim Biophys Acta. 2000 Aug. 31; 1481(1):147-55. 2000). The proteinsequence suggests that it spans the membrane once, with its NH₂ terminusoriented toward the lumen of the ER or the extracellular space. The NH₂terminus contains an EF-hand domain, and functions as the Ca²⁺ sensor inthe ER. The protein also contains protein-protein interaction domains,notably coiled-coiled domains in the cytoplasm and a sterile motif (SAM)in the ER (or extracellular space), both near the predictedtransmembrane domain. STIM1 can oligomerize and thus the protein in theER and plasma membrane could interact bridging the two (Roos, J. et al.J. Cell Biol. 169, 435-445 (2005)).

Total internal reflection fluorescence (TIRF) and confocal microscopyreveal that STIM1 is distributed throughout the ER when Ca²⁺ stores arefull, but redistributes into discrete puncta near the plasma membrane onstore depletion. Although the redistribution of STIM1 into junctional ERregions is slow (Liou, J. et al. Curr. Biol. 15, 1235-1241 (2005);Zhang, S. L. et al. Nature 437, 902-905 (2005), it does precede theopening of CRAC channels by several seconds (Wu et al., J. Cell Biol.174, 803-813 (2006)) and is therefore rapid enough to be an essentialstep in the activation of CRAC channels.

It has been suggested that store depletion causes the insertion of STIM1into the plasma membrane where it may control store operated calciumentry through the CRAC channels (Zhang, S. L. et al. Nature 437, 902-905(2005); Spassova, M. A. et al. Proc. Natl Acad. Sci. USA 103, 4040-4045(2006)).

The critical evidence for STIM1 as the Ca²⁺ sensor for SOCE is thatmutation of predicted Ca²⁺-binding residues of the EF hand structuralmotif, expected to reduce its affinity for Ca²⁺ and hence mimic thestore-depleted state, causes STIM1 to redistribute spontaneously intopuncta and trigger constitutive Ca²⁺ influx through SOCs even whenstores are full (Spassova, M. A. et al. Proc. Natl Acad. Sci. USA 103,4040-4045 (2006); Liou, J. et al. Curr. Biol. 15, 1235-1241 (2005)).

Orai Proteins

Orai1 (also known as CRACM1) is a widely expressed, 33 kDa plasmamembrane protein with 4 transmembrane domains and a lack of significantsequence homology to other ion channels (Vig, M. et al. Science312,1220-1223 (2006); Zhang, S. L. et al. Proc. Natl Acad. Sci. USA 103,9357-9362 (2006)).

Studies of T cells from human patients with a severe combinedimmunodeficiency (SCID) syndrome, in which T cell receptor engagement orstore depletion failed to activate Ca²⁺ entry, was shown to be due to asingle point mutation in Orai1 (Feske, S. et al. Nature 441,179-185(2006)).

Other mammalian Orai homologues exist, e.g. Orai2 and Orai3, howevertheir function is not clearly defined. Orai2 and Orai3 can exhibit SOCchannel activity when overexpressed with STIM1 in HEK cells (Mercer, J.C. et al. J. Biol. Chem. 281,24979-24990 (2006)).

Evidence that Orai1 contributes to the CRAC channel pore was obtained byOrai1 mutagenesis studies. Selectivity of the CRAC channel for Ca²⁺ ionswas shown by mutations at either Glu 106 or Glu 190, which weaken theability of Ca²⁺ binding in order block permeation of monovalent cations(similar to mechanisms described for voltage-gated Ca²⁺ channels)(Yeromin, A. V. et al. Nature 443,226-229 (2006); Vig, M. et al. Curr.Biol. 16,2073-2079 (2006); Prakriya, M. et al. Nature 443,230-233(2006)).

Neutralizing the charge on a pair of aspartates in the I-II loop (Asp110 and Asp 112) reduces block by Gd³⁺ and block of outward current byextracellular Ca²⁺, indicating that these negatively charged sites maypromote accumulation of polyvalent cations near the mouth of the pore.

Currents observed through overexpression of Orai1 closely resembleI_(CRAC), and the fact that Orai1 can form multimers (Yeromin, A. V. etal. Nature 443,226-229 (2006); Vig, M. et al. Curr. Biol. 16,2073-2079(2006); Prakriya, M. et al. Nature 443,230-233 (2006)), it is likelythat the native CRAC channel is either a multimer of Orail alone or incombination with the closely related subunits Orai2 and/or Orai3.

Functional Store Operated Calcium Channels

The characterization of SOC channels has been largely obtained by onetype of SOC channel, the CRAC channel. CRAC channel activity istriggered by the loss of Ca²⁺ from the ER lumen, which is coupled to theopening of CRAC channels in the plasma membrane through the actions ofSTIM1 and Orail. Depletion of Ca²⁺ is sensed by STIM1, causing it toaccumulate in junctional ER adjacent to the plasma membrane. In aTIRF-based Ca²⁺-imaging study to map the locations of open CRACchannels, [Ca^(2+]) _(i) elevations were seen to co-localize with STIM1puncta, showing directly that CRAC channels open only in extremeproximity to these sites (Luik, et al., J. Cell Biol. 174,815-825(2006)).

In cells co-expressing both STIM1 and Orai1, store depletion causesOrai1 itself to move from a dispersed distribution to accumulate in theplasma membrane directly opposite STIM1, enabling STIM1 to activate thechannel (Luik, et al., J. Cell Biol. 174, 815-825 (2006); Xu, P. et al.Biochem. Biophys. Res. Commun. 350, 969-976 (2006)). Thus, CRAC channelsare formed by apposed clusters of STIM1 in the ER and Orail in theplasma membrane, separated by a narrow gap of cytosol. The junctionalgap (about 10-25 nm) may be small enough to permit protein-proteininteractions. This is supported by the fact that overexpressed STIM1 andOrai1 can be co-immunoprecipitated (Yeromin, A. V. et al. Nature 443,226-229 (2006); Vig, M. et al. Curr. Biol. 16, 2073-2079 (2006)).

Thus, STIM1 and Orail interact either directly or as members of amultiprotein complex. Support for this was observed when the expressionof the cytosolic portion of STIM1 by itself was sufficient to activateCRAC channels in one study (Huang, G. N. et al. Nature Cell Biol. 8,1003-1010 (2006)), and the effects of deleting the ERM/coiled-coil andother C-terminal domains suggest roles in STIM1 clustering and SOCchannel activation (Baba, Y. et al. Proc. Natl Acad. Sci. USA 103,16704-16709 (2006)). On the luminal side of STIM1, the isolated EF-SAMregion forms dimers and higher-order multimers on removal of Ca²⁺ invitro, indicating that STIM1 oligomerization may be an early step instore operated calcium activation (Stathopulos, et al., J. Biol. Chem.281, 35855-35862 (2006)).

Compounds of Formula (I), (II), (IIA) and (IIB) described hereinmodulate intracellular calcium, such as, inhibition or reduction of SOCEand/or I_(CRAC). The modulation by compounds of Formula (I), (II), (IIA)and (IIB) may result from a variety of effects, such as, but not limitedto, binding to a protein, interaction with a protein, or modulation ofinteractions, activities, levels or any physical, structural or otherproperty of a protein involved in modulating intracellular calcium (e.g.a STIM protein and/or Orai protein).

For example, methods for assessing binding or interaction of a testagent with a protein involved in modulating intracellular calciuminclude NMR, mass spectroscopy, fluorescence spectroscopy, scintillationproximity assays, surface plasmon resonance assays and others. Examplesof methods for assessing modulation of interactions, activities, levelsor any physical, structural or other property of a protein involved inmodulating intracellular calcium include, but are not limited to, FRETassays to assess effects on protein interactions, NMR, X-raycrystallography and circular dichroism to assess effects on proteininteractions and on physical and structural properties of a protein, andactivity assays suitable for assessing a particular activity of aprotein.

Monitoring or Assessing Effects on Intracellular Calcium

In monitoring or assessing the effect of a compound of Formula (I),(II), (IIA) or (IIB) on intracellular calcium in any of thescreening/identification methods described herein or known in the art, adirect or indirect evaluation or measurement of cellular (includingcytosolic and intracellular organelle or compartment) calcium and/ormovement of ions into, within or out of a cell, organelle, calcium storeor portions thereof (e.g., a membrane) can be conducted. A variety ofmethods are described herein and/or known in the art for evaluatingcalcium levels and ion movements or flux. The particular method used andthe conditions employed can depend on whether a particular aspect ofintracellular calcium is being monitored or assessed. For example, asdescribed herein, reagents and conditions are known, and can be used,for specifically evaluating store-operated calcium entry, restingcytosolic calcium levels, calcium buffering and calcium levels anduptake by or release from intracellular organelles and calcium stores.The effect of a compound of Formula (I), (II), (IIA) or (IIB) onintracellular calcium can be monitored or assessed using, for example, acell, an intracellular organelle or calcium storage compartment, amembrane (including, e.g., a detached membrane patch or a lipid bilayer)or a cell-free assay system (e.g., outside-out membrane vesicle).Generally, some aspect of intracellular calcium is monitored or assessedin the presence of test agent and compared to a control, e.g.,intracellular calcium in the absence of test agent.

Methods of Modulating Intracellular Calcium

Modulation of intracellular calcium can be any alteration or adjustmentin intracellular calcium including but not limited to alteration ofcalcium concentration or level in the cytoplasm and/or intracellularcalcium storage organelles, e.g., endoplasmic reticulum, alteration inthe movement of calcium into, out of and within a cell or intracellularcalcium store or organelle, alteration in the location of calcium withina cell, and alteration of the kinetics, or other properties, of calciumfluxes into, out of and within cells. In particular embodiments,intracellular calcium modulation can involve alteration or adjustment,e.g. reduction or inhibition, of store-operated calcium entry, cytosoliccalcium buffering, calcium levels in or movement of calcium into, out ofor within an intracellular calcium store or organelle, and/or basal orresting cytosolic calcium levels. In some embodiments, modulation ofintracellular calcium can involve an alteration or adjustment inreceptor-mediated ion (e.g., calcium) movement, secondmessenger-operated ion (e.g., calcium) movement, calcium influx into orefflux out of a cell, and/or ion (e.g., calcium) uptake into or releasefrom intracellular compartments, including, for example, endosomes andlysosomes.

In one aspect, compounds described herein modulate intracellularcalcium, such as but not limited to, modulation (e.g. reduction orinhibition) of SOC channel activity, such as inhibition of CRAC channelactivity (e g inhibition of I_(CRAC), inhibition of SOCE), in an immunesystem cell (e.g., a lymphocyte, white blood cell, T cell, B cell), afibroblast (or a cell derived from a fibroblast), or an epidermal,dermal or skin cell (e.g., a keratinocyte). The step of modulating oneor more proteins involved in modulating intracellular calcium (e.g. aSTIM protein and/or Orai protein) can involve, for example, reducing thelevel, expression of, an activity of, function of and/or molecularinteractions of a protein. For instance, if a cell exhibits an increasein calcium levels or lack of regulation of an aspect of intracellularcalcium modulation, e.g., store-operated calcium entry, then modulatingmay involve reducing the level of, expression of, an activity orfunction of, or a molecular interaction of a protein, e.g. a STIMprotein and/or Orai protein.

Treatment Methods

Presented herein is a method of modulating store-operated calcium (SOC)channel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof:

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R² is aryl, benzothienyl, benzofuranyl, or -CH₂—H₂-phenyl; wherein    R² is optionally substituted with 1 or 2 substituents independently    selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,    C₁-C₆alkyl, C₃-C₆cycloalkyl, C_(l)-C₆heteroalkyl, C₁-C₆haloalkyl,    tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,    —S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸,    —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂,    —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   R⁴ is an aryl, optionally substituted with 1 or 2 substituents    independently selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

In one embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I),wherein the contacting occurs in vitro.

In another embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I)

-   -   wherein the contacting occurs in vivo.

In yet another embodiment is a method of modulating store-operatedcalcium channel activity comprising contacting the store-operatedcalcium (SOC) channel complex, or portion thereof, with a compound ofFormula (I), wherein the compound of Formula (I) modulates an activityof, modulates an interaction of, or modulates the level of, or binds to,or interacts with at least one portion of the store operated calciumchannel complex selected from stromal interaction molecules (STIM)family of proteins.

In a further embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I),wherein the compound of Formula (I) modulates an activity of, modulatesan interaction of, or modulates the level of, or binds to, or interactswith at least one portion of STIM1 or STIM2.

In another embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I),wherein modulating store operated calcium channel activity with acompound of Formula (I) inhibits store-operated calcium entry (SOCE).

In yet another embodiment is a method of modulating store-operatedcalcium channel activity comprising contacting the store-operatedcalcium (SOC) channel complex, or portion thereof, with a compound ofFormula (I), wherein the store operated calcium channel complex iscalcium-release activated calcium (CRAC) channel complex.

In a further embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I),wherein modulating calcium release activated calcium (CRAC) activitywith a compound of Formula (I) inhibits the electrophysiological current(I_(CRAC)) directly associated with activated CRAC channels.

In yet a further embodiment is a method of modulating store-operatedcalcium channel activity comprising contacting the store-operatedcalcium (SOC) channel complex, or portion thereof, with a compound ofFormula (I), wherein R¹ is hydrogen or C₁-C₆alkyl.

In one embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I),wherein R⁴ is selected from phenyl; 2-fluorophenyl; 3-fluorophenyl;4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl;2,4-dichlorophenyl; 2,3-dichlorophenyl; 3,4-dichlorophenyl;3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl; 4-bromophenyl;2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; 2-trifluoromethylphenyl;3-trifluoromethylphenyl; and 4-trifluoromethylphenyl.

In another embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I),wherein R² is selected from phenyl; 2-fluorophenyl; 3-fluorophenyl;4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl;2,4-dichlorophenyl; 2,3-dichlorophenyl; 3,4-dichlorophenyl;3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl; 4-bromophenyl;2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; and benzothien-2-yl.

In yet another embodiment is a method of modulating store-operatedcalcium channel activity comprising contacting the store-operatedcalcium (SOC) channel complex, or portion thereof, with a compound ofFormula (I), wherein R¹ is hydrogen, methyl, ethyl, n-propyl, oriso-propyl.

In a further embodiment is a method of modulating store-operated calciumchannel activity comprising contacting the store-operated calcium (SOC)channel complex, or portion thereof, with a compound of Formula (I),wherein R² is selected from phenyl, 3-fluorophenyl; 4-fluorophenyl;4-chlorophenyl; 4-bromophenyl; 4-iodophenyl; 3-methylphenyl;3-methylphenyl; and benzothien-2-yl.

In yet a further embodiment is a method of modulating store-operatedcalcium channel activity comprising contacting the store-operatedcalcium (SOC) channel complex, or portion thereof, with a compound ofFormula (I), wherein R⁴ is selected from phenyl; 4-fluorophenyl;2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl; 2,4-dichlorophenyl;3,4-dichlorophenyl; 3,5-dichlorophenyl; 2-bromophenyl; 4-bromophenyl;4-methylphenyl; 3,4-dimethylphenyl; and 4-trifluoromethylphenyl.

Also presented herein is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof:

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R² is aryl, benzothienyl, benzofuranyl, or —CH₂CH₂-phenyl; wherein    R² is optionally substituted with 1 or 2 substituents independently    selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,    C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,    tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,    —S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸,    —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂,    —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

In one embodiment is a method of modulating calcium release activatedcalcium channel (CRAC) activity in a mammal comprising administering acompound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the compound of Formula (I) modulates anactivity of, modulates an interaction of, or modulates the level of, orbinds to, or interacts with at least one component of the calciumrelease activated (CRAC) channel complex selected from stromalinteraction molecules (STIM) family of proteins.

In another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein the compound of Formula (I) modulatesan activity of, modulates an interaction of, or modulates the level of,or binds to, or interacts with STIM1 or STIM2.

In yet another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein modulating calcium release activatedcalcium (CRAC) channel activity with a compound of Formula (I) inhibitsstore-operated calcium entry (SOCE).

In a further embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein modulating calcium release activatedcalcium (CRAC) channel activity with a compound of Formula (I) inhibitsthe electrophysiological current (I_(CRAC)) directly associated withactivated CRAC channels.

In yet a further embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein the compound of Formula (I) inhibitsSOCE with an IC₅₀ below 10 μM.

In another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein the compound of Formula (I) inhibitselectrophysiological current (I_(CRAC)) directly associated withactivated CRAC channels at a concentration below 10 μM.

In yet another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R⁴ is phenyl, optionally substitutedwith 1 or 2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃,—OH, —OR⁸, —OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl.

In a further embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R¹ is hydrogen or C₁-C₆alkyl.

In one embodiment is a method of modulating calcium release activatedcalcium channel (CRAC) activity in a mammal comprising administering acompound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein R² is phenyl or benzothienyl; wherein R² isoptionally substituted with 1 or 2 substituents independently selectedfrom F, Cl, Br, I, —CN, —NO₂, —OH, _(—CF) ₃, —OCF₃, —OR⁸, C₁-C₆alkyl,C₁-C₆heteroalkyl, and C₁-C₆haloalkyl.

In yet a further embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R¹ is H, methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, iso-butyl, n-pentyl, or hexyl.

In one embodiment is a method of modulating calcium release activatedcalcium channel (CRAC) activity in a mammal comprising administering acompound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein R¹ is H, methyl, or ethyl.

In another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R⁴ is phenyl, optionally substitutedwith 1 or 2 substituents selected from F, Cl, Br, I, —CN, —CF₃, —OH,—OMe, —OCF₃, methyl, and ethyl.

In yet another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R² is phenyl or benzothienyl; whereinR² is optionally substituted with 1 or 2 substituents independentlyselected from F, Cl, Br, I, —CN, —OH, —CF₃, —OCF₃, —OMe, methyl, andethyl.

In a further embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R⁴ is selected from phenyl;2-fluorophenyl; 3-fluorophenyl; 4-fluorophenyl; 2-chlorophenyl;3-chlorophenyl; 4-chlorophenyl; 2,4-dichlorophenyl; 2,3-dichlorophenyl;3,4-dichlorophenyl; 3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl;4-bromophenyl; 2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; 2-trifluoromethylphenyl;3-trifluoromethylphenyl; and 4-trifluoromethylphenyl.

In another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R² is selected from phenyl;2-fluorophenyl; 3-fluorophenyl; 4-fluorophenyl; 2-chlorophenyl;3-chlorophenyl; 4-chlorophenyl; 2,4-dichlorophenyl; 2,3-dichlorophenyl;3,4-dichlorophenyl; 3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl;4-bromophenyl; 2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; and benzothien-2-yl.

In yet another embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R² is selected from phenyl,3-fluorophenyl; 4-fluorophenyl; 4-chlorophenyl; 4-bromophenyl;4-iodophenyl; 3-methylphenyl; 3-methylphenyl; and benzothien-2-yl.

In a further embodiment is a method of modulating calcium releaseactivated calcium channel (CRAC) activity in a mammal comprisingadministering a compound of Formula (I), or pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof wherein R⁴ is selected from phenyl;4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl;2,4-dichlorophenyl; 3,4-dichlorophenyl; 3,5-dichlorophenyl;2-bromophenyl; 4-bromophenyl; 4-methylphenyl; 3,4-dimethylphenyl; and4-trifluoromethylphenyl.

In one aspect is a method of treating a disease, disorder or conditionin a mammal that would benefit from inhibition of store operated calciumchannel activity comprising administering to the mammal a compound ofFormula (I), or pharmaceutically acceptable salt, pharmaceuticallyacceptable solvate, or pharmaceutically acceptable prodrug thereof:

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R² is aryl, benzothienyl, benzofuranyl, or —CH₂CH₂-phenyl; wherein    R² is optionally substituted with 1 or 2 substituents independently    selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,    C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,    tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,    —S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸,    —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂,    —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

In one embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the compound of Formula (I) modulates theactivity of, modulates an interaction of, or binds to, or interacts witha mammalian STIM1 protein, or a mammalian STIM2 protein.

In another embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the disease, disorder or condition in a mammalis selected from diseases/disorders involving inflammation,glomerulonephritis, uveitis, hepatic diseases or disorders, renaldiseases or disorders, chronic obstructive pulmonary disease, rheumatoidarthritis, psoriasis, inflammatory bowel disease, vasculitis,dermatitis, osteoarthritis, inflammatory muscle disease, allergicrhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis,eczema, organ transplant rejection, allogeneic or xenogeneictransplantation, graft rejection, graft-versus-host disease, lupuserythematosus, type I diabetes, pulmonary fibrosis, dermatomyositis,thyroiditis, myasthenia gravis, autoimmune hemolytic anemia, cysticfibrosis, chronic relapsing hepatitis, primary biliary cirrhosis,allergic conjunctivitis, hepatitis and atopic dermatitis, asthma,multiple sclerosis, Sjogren's syndrome, and autoimmune diseases ordisorders.

In yet another embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the disease, disorder or condition is rheumatoidarthritis.

In a further embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the disease, disorder or condition is psoriasis.

In one embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the disease, disorder or condition is isinflammatory bowel disease.

In a further embodiment the inflammatory bowel disease is ulcerativecolitis.

In a further embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the disease, disorder or condition is organtransplant rejection.

In a further embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein the disease, disorder or condition is multiplesclerosis.

In yet a further embodiment is a method of treating a disease, disorderor condition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof further comprising administering to the mammal a secondtherapeutic agent.

In another embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein the second therapeutic agent is selected fromimmunosuppressants, glucocorticoids, non-steroidal anti-inflammatorydrugs, Cox-2-specific inhibitors, leflunomide, gold thioglucose, goldthiomalate, aurofin, sulfasalazine, hydroxychloroquinine, minocycline,anti-TNF-α agents, abatacept, anakinra, interferon-β, interferon-γ,interleukin-2, allergy vaccines, antihistamines, antileukotrienes,beta-agonists, theophylline, and anticholinergics.

In yet another embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein the second therapeutic agent is selected fromtacrolimus, cyclosporin, rapamicin, methotrexate , cyclophosphamide,azathioprine, mercaptopurine, mycophenolate, or FTY720, prednisone,cortisone acetate, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate, aldosterone, aspirin, salicylic acid,gentisic acid, choline magnesium salicylate, choline salicylate, cholinemagnesium salicylate, choline salicylate, magnesium salicylate, sodiumsalicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium,fluorobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac, ketorolactromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin,sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid,piroxicam, meloxicam, celecoxib, rofecoxib, valdecoxib, parecoxib,etoricoxib, lumiracoxib, CS-502, JTE-522, L-745,337 and NS398,leflunomide, gold thioglucose, gold thiomalate, aurofin, sulfasalazine,hydroxychloroquinine, minocycline, infliximab, etanercept, adalimumab,abatacept, anakinra, interferon-β, interferon-γ, interleukin-2, allergyvaccines, antihistamines, antileukotrienes, beta-agonists, theophylline,and anticholinergics.

In a further embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein R¹ is H, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, n-pentyl, or hexyl.

In another embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof wherein R¹ is H, methyl, or ethyl.

In one embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein R⁴ is a phenyl, optionally substituted with 1or 2 substituents selected from F, Cl, Br, I, —CN, —CF₃, —OH, —OMe,—OCF₃, methyl, and ethyl.

In yet a further embodiment is a method of treating a disease, disorderor condition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein R² is phenyl or benzothienyl; wherein R² isoptionally substituted with 1 or 2 substituents independently selectedfrom F, Cl, Br, I, —CN, —OH, —CF₃, —OCF₃, —OMe, methyl, and ethyl.

In one embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein R⁴ is selected from phenyl; 2-fluorophenyl;3-fluorophenyl; 4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl;4-chlorophenyl; 2,4-dichlorophenyl; 2,3-dichlorophenyl;3,4-dichlorophenyl; 3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl;4-bromophenyl; 2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; 2-trifluoromethylphenyl;3-trifluoromethylphenyl; and 4-trifluoromethylphenyl.

In a further embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein R² is selected from phenyl; 2-fluorophenyl;3-fluorophenyl; 4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl;4-chlorophenyl; 2,4-dichlorophenyl; 2,3-dichlorophenyl;3,4-dichlorophenyl; 3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl;4-bromophenyl; 2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; and benzothien-2-yl.

In another embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein R² is selected from phenyl, 3-fluorophenyl;4-fluorophenyl; 4-chlorophenyl; 4-bromophenyl; 4-iodophenyl;3-methylphenyl; 3-methylphenyl; and benzothien-2-yl.

In yet another embodiment is a method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof, wherein R⁴ is selected from phenyl; 4-fluorophenyl;2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl; 2,4-dichlorophenyl;3,4-dichlorophenyl; 3,5-dichlorophenyl; 2-bromophenyl; 4-bromophenyl;4-methylphenyl; 3,4-dimethylphenyl; and 4-trifluoromethylphenyl.

Also described herein is a method of inhibiting store-operated calciumentry (SOCE) activation of nuclear factor of activated T cells (NFAT) ina mammal comprising administering a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof:

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R² is aryl, benzothienyl, benzofuranyl, or -CH₂CH₂-phenyl; wherein    R² is optionally substituted with 1 or 2 substituents independently    selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,    C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,    tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,    —S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸,    —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂,    —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

In one embodiment is a method of inhibiting store-operated calcium entry(SOCE) activation of nuclear factor of activated T cells (NFAT) in amammal comprising administering a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof, wherein the compound ofFormula (I) modulates an interaction of, or modulates the level of, orbinds to, or interacts with a mammalian STIM1 protein, or a mammalianSTIM2 protein.

In another aspect is a method of decreasing cytokine expression byinhibiting the store-operated calcium entry activation of NFAT in amammal comprising administering a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof:

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R² is aryl, benzothienyl, benzofuranyl, or —CH₂CH₂-phenyl; wherein    R² is optionally substituted with 1 or 2 substituents independently    selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,    C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,    tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,    —S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸,    —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂,    —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

In another embodiment is a method of decreasing cytokine expression byinhibiting the store-operated calcium entry activation of NFAT in amammal comprising administering a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof wherein the compound ofFormula (I) modulates an interaction of, or modulates the level of, orbinds to, or interacts with a mammalian STIM1 protein or a mammalianSTIM2 protein.

In yet another embodiment is a method of decreasing cytokine expressionby inhibiting the store-operated calcium entry activation of NFAT in amammal comprising administering a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereofwherein the cytokine isselected from IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1α, IL-1β, IL-1 RA,granulocyte colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), oncostatin M, erythropoietin,leukemia inhibitory factor (LIF), interferons, gamma-interferon (γ-IFN),B7.1 (CD80), B7.2 (B70, CD86), TNF-α, TNF-β, LT-β, CD40 ligand, Fasligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail, and migrationinhibitory factor (MIF).

Compounds

Compounds described herein modulate intracellular calcium and may beused in the treatment of diseases or conditions where modulation ofintracellular calcium has a beneficial effect. In one embodiment,compounds described herein inhibit store operated calcium entry. In oneembodiment, compounds of Formula (I), (II), (IIA) or (IIB) interrupt theassembly of SOCE units. In another embodiment, compounds of Formula (I),(II), (IIA) or (IIB) alter the functional interactions of proteins thatform store operated calcium channel complexes. In one embodiment,compounds of Formula (I), (II), (IIA) or (IIB) alter the functionalinteractions of STIM1 with Orail. In other embodiments, compounds ofFormula (I), (II), (IIA) or (IIB) are SOC channel pore blockers. Inother embodiments, compounds of Formula (I), (II), (IIA) or (IIB) areCRAC channel pore blockers.

In one aspect, compounds of Formula (I), (II), (IIA) or (IIB) inhibitthe electrophysiological current (I_(SOC)) directly associated withactivated SOC channels. In one aspect, compounds of Formula (I), (II),(IIA) or (IIB) inhibit the electrophysiological current (_(CRAC))directly associated with activated CRAC channels.

The diseases or disorders that may benefit from modulation ofintracellular calcium include, but are not limited to, an immunesystem-related disease (e.g., an autoimmune disease), a disease ordisorder involving inflammation (e.g., asthma, chronic obstructivepulmonary disease, rheumatoid arthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases, multiple sclerosis, anddisorders of the immune system), cancer or other proliferative disease,kidney disease and liver disease. In one aspect, compounds describedherein may be used as immunosuppresants to prevent transplant graftrejections, allogeneic or xenogeneic transplantation rejection (organ,bone marrow, stem cells, other cells and tissues), graft-versus-hostdisease. Transplant graft rejections can result from tissue or organtransplants. Graft-versus-host disease can result from bone marrow orstem cell transplantation.

Compounds described herein modulate an activity of, modulate aninteraction of, or binds to, or interacts with at least one portion of aprotein in the store operated calcium channel complex. In oneembodiment, compounds described herein modulate an activity of, modulatean interaction of, or binds to, or interacts with at least one portionof a protein in the calcium release activated calcium channel complex.In one aspect, compounds described herein reduce the level of functionalstore operated calcium channel complexes. In one aspect, compoundsdescribed herein reduce the level of activated store operated calciumchannel complexes. In one aspect, store operated calcium channelcomplexes are calcium release activated calcium channel complexes.

Compounds described herein for treatment of a disease or disorder, whenadministered to a subject having a disease or disorder effectivelyreduces, ameliorates or eliminates a symptom or manifestation of thedisease or disorder. Compounds described herein can also be administeredto a subject predisposed to a disease or disorder who does not yetmanifest a symptom of the disease or disorder, prevents or delaysdevelopment of the symptoms. The agent can have such effects alone or incombination with other agents, or may function to enhance a therapeuticeffect of another agent.

Compounds described herein, pharmaceutically acceptable salts,pharmaceutically acceptable prodrugs, or pharmaceutically acceptablesolvates thereof, modulate intracellular calcium, and may be used totreat patients where modulation of intracellular calcium providesbenefit.

In one aspect, described herein is a compound of Formula (I), orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof:

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R² is aryl, benzothienyl, benzofuranyl, or —CH₂CH₂-phenyl; wherein    R² is optionally substituted with 1 or 2 substituents independently    selected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸, C₁-C₆    alkyl, C₃-C₆cycloalkyl, C₁ -C₆heteroalkyl, C₁-C₆haloalkyl,    tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,    —S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸,    —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂,    —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl.

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, R¹ is hydrogen or C₁-C₆alkyl. In other embodiments, R¹ isH, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,t-butyl, n-pentyl, or hexyl. In yet other embodiments, R¹ is H, methyl,or ethyl. In some embodiments, R¹ is H. In one embodiment, the carboxylmoiety of the thiophene core is replaced with a carboxylic acidbioisostere.

In some embodiments, R² is phenyl, naphthyl, or benzothienyl; wherein R²is optionally substituted with 1 or 2 substituents independentlyselected from F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸,C₁-C₆alkyl, C₁-C₆heteroalkyl, and C₁-C₆haloalkyl.

In some embodiments, R² is optionally substituted with 1 or 2substituents selected from among F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃,—OCF₃, —OR⁸, C₁-C₆alkyl, C₁-C₆haloalkyl, tetrazolyl,C₂-C₆heterocycloalkyl, phenyl, —N(R⁹)₂, —CO₂R⁹, —C(═O)R⁸. In someembodiments, R² is optionally substituted with 1 or 2 substituentsselected from among hydrogen, F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃,—OMe, —OEt, -OiPr, methyl, ethyl, n-propyl, i-propyl, n-butyl,sec-butyl, t-butyl, phenyl, —NH₂, —N(Me)₂, —CO₂H, —CO₂Me, and —CO₂Et. Inother embodiments, R² is optionally substituted with 1 or 2 substituentsselected from among —NH(CO)CH₃, -propyl, —CF₃, methyl, ethyl, —SO₂CH₃,—CN, or —OCH₃.

In some embodiments, R² is phenyl or benzothienyl; wherein R² isoptionally substituted with 1 or 2 substituents independently selectedfrom F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸, C₁-C₆alkyl,C₁-C₆heteroalkyl, and C₁-C₆haloalkyl. In other embodiments, R² is phenylor benzothienyl; wherein R² is optionally substituted with 1 or 2substituents independently selected from F, Cl, Br, I, —CN, —OH, —CF₃,—OCF₃, —OMe, methyl, ethyl, isopropyl, and t-butyl. In some embodiments,R² is selected from phenyl; 2-fluorophenyl; 3-fluorophenyl;4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl;2,4-dichlorophenyl; 2,3-dichlorophenyl; 3,4-dichlorophenyl;3,5-dichlorophenyl; 2-bromophenyl; 3-bromophenyl; 4-bromophenyl;2-iodophenyl; 3-iodophenyl; 4-iodophenyl; 2-methylphenyl;3-methylphenyl; 4-methylphenyl; 2,4-dimethylphenyl; 2,3-dimethylphenyl;3,4-dimethylphenyl; 3,5-dimethylphenyl; and benzothien-2-yl. In otherembodiments, R² is selected from phenyl, 3-fluorophenyl; 4-fluorophenyl;4-chlorophenyl; 4-bromophenyl; 4-iodophenyl; 3-methylphenyl;3-methylphenyl; and benzothien-2-yl.

In some embodiments, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl. In other embodiments, R⁴ is a phenyl, optionallysubstituted with 1 or 2 substituents selected from F, Cl, Br, I, —CN,—CF₃, —OH, —OMe, —OCF₃, methyl, and ethyl. In yet other embodiments, R⁴is selected from phenyl; 2-fluorophenyl; 3-fluorophenyl; 4-fluorophenyl;2-chlorophenyl; 3-chlorophenyl; 4-chlorophenyl; 2,4-dichlorophenyl;2,3-dichlorophenyl; 3,4-dichlorophenyl; 3,5-dichlorophenyl;2-bromophenyl; 3-bromophenyl; 4-bromophenyl; 2-iodophenyl; 3-iodophenyl;4-iodophenyl; 2-methylphenyl; 3-methylphenyl; 4-methylphenyl;2,4-dimethylphenyl; 2,3-dimethylphenyl; 3,4-dimethylphenyl;3,5-dimethylphenyl; 2-trifluoromethylphenyl; 3-trifluoromethylphenyl;and 4-trifluoromethylphenyl. In yet some other embodiments, R⁴ isselected from phenyl; 4-fluorophenyl; 2-chlorophenyl; 3-chlorophenyl;4-chlorophenyl; 2,4-dichlorophenyl; 3,4-dichlorophenyl;3,5-dichlorophenyl; 2-bromophenyl; 4-bromophenyl; 4-methylphenyl;3,4-dimethylphenyl; and 4-trifluoromethylphenyl.

In one embodiment is a compound of Formula (I) wherein R⁴ is4-chlorophenyl, R¹ is hydrogen, and R² is CH₂CH₂-phenyl. In anotherembodiment is a compound of Formula (I) wherein R⁴ is3,4-dichlorophenyl, R¹ is hydrogen, and R² is CH₂CH₂-phenyl. In afurther embodiment is a compound of Formula (I) wherein R⁴ is3,5-dichlorophenyl, R¹ is hydrogen and R² is CH₂CH₂-phenyl.

Any combination of the groups described above for the various variablesis contemplated herein.

In one aspect, the compound of Formula (I) is selected from among:

2-(4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(4-fluorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(2-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(phenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-iodobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(3,5-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(4-bromobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(3 -fluorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(4-chlorobenzamido)-4-(phenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylicacid;

2-(benzothien-2-ylamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(benzothien-2-ylamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylicacid;

2-(3-methylbenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(phenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(3 -methylbenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

Methyl 2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylate;

Ethyl 2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylate;

2-(4-iodobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

4-(2,4-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylicacid;

4-(3,4-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylicacid;

4-(3,5-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(3-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(2-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(2-chloro-4-fluorobenzamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3,4-difluorobenzamido)thiophene-3-carboxylic acid;

2-(2-chloro-4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(2-fluorobenzamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(3-fluoro-4-methoxybenzamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(4-methylbenzamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(4-cyanobenzamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(4-ethylbenzamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(4-(trifluoromethyl)benzamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3-chlorophenyl)propanamido)thiophene-3-carboxylicacid; and

4-(4-chlorophenyl)-2-(3-(4-chlorophenyl)propanamido)thiophene-3-carboxylicacid; or pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof.

In another aspect, the compound of Formula (I) is selected from among:

2-(3-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(phenylpropanamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(3-fluorophenylpropanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-chlorophenylpropanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(2-chloro-4-fluorobenzamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3,4-difluorobenzamido)thiophene-3-carboxylic acid;

2-(2-chloro-4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(2-fluorobenzamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(3-fluoro-4-methoxybenzamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(4-methylbenzamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(4-cyanobenzamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(4-ethylbenzamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(4-(trifluoromethyl)benzamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3-chlorophenyl)propanamido)thiophene-3-carboxylicacid; and

4-(4-chlorophenyl)-2-(3-(4-chlorophenyl)propanamido)thiophene-3-carboxylicacid; or pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof.

In yet another aspect, the compound of Formula (I) is selected fromamong:

2-(3-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3-chlorophenyl)propanamido)thiophene-3-carboxylicacid;

2-(4-bromobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;and

2-(4-iodobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid; orpharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof.

In another aspect is a compound of Formula (I) selected from among:

or pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof.

In one aspect, described herein is a compound of Formula (II):

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl; or    pharmaceutically acceptable salt, pharmaceutically acceptable    solvate, or pharmaceutically acceptable prodrug thereof.

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, R¹ is hydrogen or C₁-C₆alkyl. In other embodiments, R¹ ishydrogen, methyl, ethyl, n-propyl, or iso-propyl. In other embodiments,R¹ is t-butyl. In yet other embodiments, R¹ is hydrogen. In oneembodiment, the carboxyl moiety of the thiophene core is replaced with acarboxylic acid bioisostere.

In some embodiments, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, -Ole,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl. In other embodiments, R⁴ is a phenyl, optionallysubstituted with 1 or 2 substituents selected from F, Cl, Br, I, —CF₃,—OH, —OCH₃, —OCF₃, methyl, and ethyl.

Further embodiments of compounds described herein include, but are notlimited to, compounds in Table 1.

TABLE 1 Representative compounds of Formula (I) and Formula (II).

Compound no. R² —R¹ —R⁴ 1 4-fluorophenyl —H 4-bromophenyl 24-fluorophenyl —H 4-chlorophenyl 3 4-fluorophenyl —H 3,4-dichlorophenyl4 4-fluorophenyl —H 4-trifluoromethylphenyl 5 4-fluorophenyl —H2-bromophenyl 6 4-fluorophenyl —H 3,4-dimethylphenyl 7 4-fluorophenyl —H2-chlorophenyl 8 4-fluorophenyl —H 2,4-dichlorophenyl 9 4-fluorophenyl—H Phenyl 10 4-fluorophenyl —H 4-methylphenyl 11 3-fluorophenyl —H4-bromophenyl 12 4-chlorophenyl —H 4-bromophenyl 13 4-iodobenzamido —H2,4-dichlorophenyl 14 benzothien-2-y1 —H 4-bromophenyl 15 3-methylphenyl—H 4-bromophenyl 16 4-bromophenyl —H 4-methylphenyl 17 4-bromophenyl —H4-chlorophenyl 18 4-bromophenyl —H 4-bromophenyl 19 4-bromophenyl —H3,5-dichlorophenyl 20 4-bromophenyl —H 3-chlorophenyl 21 4-bromophenyl—H 3,4-dimethylphenyl 22 4-bromophenyl —H 4-trifluoromethylphenyl 234-bromophenyl —H 3,4-dichlorophenyl 24 4-bromophenyl —H 2-bromophenyl 253-fluorophenyl —H 4-chlorophenyl 26 3-fluorophenyl —H 2,4-dichlorophenyl27 3-fluorophenyl —H 3,4-dimethylphenyl 28 3-fluorophenyl —H3-chlorophenyl 29 3-fluorophenyl —H 4-methylphenyl 30 3-fluorophenyl —H3,4-dichlorophenyl 31 3-fluorophenyl —H 2-bromophenyl 32 3-fluorophenyl—H 4-trifluoromethylphenyl 33 4-chlorophenyl —H 4-chlorophenyl 344-chlorophenyl —H 4-methylphenyl 35 4-chlorophenyl —H 2,4-dichlorophenyl36 4-chlorophenyl —H 4-fluorophenyl 37 4-chlorophenyl —H3,4-dichlorophenyl 38 4-chlorophenyl —H 3,4-dimethylphenyl 394-chlorophenyl —H 4-trifluoromethylphenyl 40 4-chlorophenyl —H Phenyl 414-chlorophenyl —H 2-bromophenyl 42 4-chlorophenyl —H 3-chlorophenyl 43benzothien-2-y1 —H 4-chlorophenyl 44 benzothien-2-y1 —H 4-methylphenyl45 benzothien-2-y1 —H 2,4-dichlorophenyl 46 benzothien-2-y1 —H3-chlorophenyl 47 benzothien-2-y1 —H 4-trifluoromethylphenyl 48benzothien-2-y1 —H 2-bromophenyl 49 benzothien-2-y1 —H3,4-dimethylphenyl 50 3-methylphenyl —H 4-chlorophenyl 51 3-methylphenyl—H 4-methylphenyl 52 3-methylphenyl —H phenyl 53 3-methylphenyl —H2-bromophenyl 54 3-methylphenyl —H 4-fluorophenyl 55 3-methylphenyl —H3-chlorophenyl 56 3-methylphenyl —H 4-trifluoromethylphenyl 573-methylphenyl —H 2,4-dichlorophenyl 58 3-methylphenyl —H3,4-dimethylphenyl 59 4-chlorophenyl —Me 4-fluorophenyl 604-chlorophenyl —Et 4-fluorophenyl 61 4-iodobenzamido —H 4-bromophenyl 62CH₂CH₂-phenyl —H 2,4-dichlorophenyl 63 CH₂CH₂-phenyl —H3,4-dichlorophenyl 64 CH₂CH₂-phenyl —H 3,5-dichlorophenyl 65CH₂CH₂-phenyl —H 4-chlorophenyl 66 CH₂CH₂-phenyl —H 3-chlorophenyl 67CH₂CH₂-phenyl —H 2-chlorophenyl 68 2-chloro,4- —H 4-bromophenylfluorophenyl 69 3,4difluorophenyl —H 4-bromophenyl 70 2-chloro,4- —H4-chlorophenyl fluorophenyl 71 2-fluorophenyl —H 4-bromophenyl 723-fluoro,4- —H 4-bromophenyl methoxyphenyl 73 4-methylphenyl —H4-chlorophenyl 74 4-cyanophenyl —H 4-bromophenyl 75 4-ethylphenyl —H4-chlorophenyl 76 4- —H 4-chlorophenyl trifluoromethylphenyl 77CH₂CH₂(3- —H 4-chlorophenyl fluorophenyl) 78 CH₂CH₂(3- —H 4-bromophenylfluorophenyl) 79 CH₂CH₂-(2,4- —H 4-chlorophenyl difluorophenyl) 80CH₂CH₂-phenyl —H 4-bromophenyl 81 CH₂CH₂(4- —H 4-bromophenylfluorophenyl) 82 CH₂CH₂-(3,4- —H 4-chlorophenyl difluorophenyl) 83CH₂CH₂-(2,4- —H 4-bromophenyl difluorophenyl) 84 CH₂CH₂-(3,4- —H4-bromophenyl difluorophenyl) 85 CH₂CH₂(4- —H 4-chlorophenylfluorophenyl) 86 CH₂CH₂(3- —H 4-chlorophenyl chlorophenyl) 87 CH₂CH₂(4-—H 4-chlorophenyl chlorophenyl)

Compounds in Table I are named:

2-(4-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(4-fluorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(2-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(phenyl)thiophene-3-carboxylic acid;

2-(4-fluorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-iodobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(3,5-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(4-bromobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-bromobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(3-fluorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(3 -fluorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(4-chlorobenzamido)-4-(phenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(4-chlorobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylicacid;

2-(benzothien-2-ylamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylicacid;

2-(benzothien-2-ylamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(benzothien-2-ylamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylicacid;

2-(3-methylbenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(phenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;

2-(3 -methylbenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic acid;

2-(3-methylbenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic acid;

Methyl 2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylate;

Ethyl 2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylate;

2-(4-iodobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;

4-(2,4-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylicacid;

4-(3,4-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylicacid;

4-(3,5-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(3-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(2-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(2-chloro-4-fluorobenzamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3,4-difluorobenzamido)thiophene-3-carboxylic acid;

2-(2-chloro-4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(2-fluorobenzamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(3-fluoro-4-methoxybenzamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(4-methylbenzamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(4-cyanobenzamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(4-ethylbenzamido)thiophene-3-carboxylic acid;

4-(4-chlorophenyl)-2-(4-(trifluoromethyl)benzamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic acid;

4-(4-bromophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-bromophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylicacid;

4-(4-chlorophenyl)-2-(3-(3-chlorophenyl)propanamido)thiophene-3-carboxylicacid; and

4-(4-chlorophenyl)-2-(3-(4-chlorophenyl)propanamido)thiophene-3-carboxylicacid.

In one aspect, described herein is a compound of Formula (II):

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;-   each R⁹ is independently selected from H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl; or    pharmaceutically acceptable salt, pharmaceutically acceptable    solvate, or pharmaceutically acceptable prodrug thereof.

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, R¹ is hydrogen or C₁-C₆alkyl. In other embodiments, R¹ ishydrogen, methyl, ethyl, n-propyl, or iso-propyl. In yet otherembodiments, R¹ is hydrogen. In one embodiment, the carboxyl moiety ofthe thiophene core is replaced with a carboxylic acid bioisostere.

In some embodiments, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl. In other embodiments, R⁴ is a phenyl, optionallysubstituted with 1 or 2 substituents selected from F, Cl, Br, I, —CF₃,—OH, —OCH₃, —OCF₃, methyl, and ethyl.

In one embodiment, R¹ is hydrogen or C₁-C₆alkyl. In yet another aspect,R¹ is hydrogen, methyl, ethyl, n-propyl, or iso-propyl.

In another embodiment, R⁴ is a phenyl, optionally substituted with 1 or2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyl, C₁-C₆heteroalkyl, andC₁-C₆haloalkyl.

In one embodiment, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CF₃, —OH, —OCH₃, —OCF₃,methyl, and ethyl.

In a further embodiment, the compound of Formula (IIA) is

In one aspect, described herein is a compound of Formula (IIB):

wherein:

-   R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl;-   R⁴ is an aryl, wherein the aryl is optionally substituted with 1 or    2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH,    —OR⁸, —OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fluoroalkyl,    C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl,    phenyl, —NHS(═O)₂R⁸, S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸,    —S(═O)₂NHC(═O)R⁹, N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸,    —OC(═O)R⁸, —C(═O)N(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸;-   each R⁸ is independently selected from C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₈cycloalkyl, phenyl, and benzyl;    -   each R⁹ is independently selected from H, C₁-C₆alkyl,        C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl; or        pharmaceutically acceptable salt, pharmaceutically acceptable        solvate, or pharmaceutically acceptable prodrug thereof.

In one embodiment, R¹ is hydrogen or C₁-C₆alkyl. In yet another aspect,R¹ is hydrogen, methyl, ethyl, n-propyl, or iso-propyl. In oneembodiment, the carboxyl moiety of the thiophene core is replaced with acarboxylic acid bioisostere.

In another embodiment, R⁴ is a phenyl, optionally substituted with 1 or2 substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₁-C₆fluoroalkyk C₁-C₆heteroalkyl, andC₁-C₆haloalkyl.

In one embodiment, R⁴ is a phenyl, optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CF₃, —OH, —OCH₃, —OCF₃,methyl, and ethyl.

In a further aspect, the compound of Formula (IIB) is selected fromamong:

Throughout the specification, groups and substituents thereof can bechosen by one skilled in the field to provide stable moieties andcompounds.

Further Forms of Compounds

The compounds described herein may in some cases exist as diastereomers,enantiomers, or other stereoisomeric forms. The compounds presentedherein include all diastereomeric, enantiomeric, and epimeric forms aswell as the appropriate mixtures thereof. Separation of stereoisomersmay be performed by chromatography or by the forming diastereomeric andseparation by recrystallization, or chromatography, or any combinationthereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers,Racemates and Resolutions”, John Wiley And Sons, Inc., 1981, hereinincorporated by reference for this disclosure). Stereoisomers may alsobe obtained by stereoselective synthesis.

In some situations, compounds may exist as tautomers. All tautomers areincluded within the formulas described herein.

The methods and compositions described herein include the use ofamorphous forms as well as crystalline forms (also known as polymorphs).The compounds described herein may be in the form of pharmaceuticallyacceptable salts. As well, active metabolites of these compounds havingthe same type of activity are included in the scope of the presentdisclosure. In addition, the compounds described herein can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms of thecompounds presented herein are also considered to be disclosed herein.

In some embodiments, compounds described herein may be prepared asprodrugs. A “prodrug” refers to an agent that is converted into theparent drug in vivo. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. An example, withoutlimitation, of a prodrug would be a compound described herein, which isadministered as an ester (the “prodrug”) to facilitate transmittalacross a cell membrane where water solubility is detrimental to mobilitybut which then is metabolically hydrolyzed to the carboxylic acid, theactive entity, once inside the cell where water-solubility isbeneficial. A further example of a prodrug might be a short peptide(polyaminoacid) bonded to an acid group where the peptide is metabolizedto reveal the active moiety. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically active form of the compound. Incertain embodiments, a prodrug is enzymatically metabolized by one ormore steps or processes to the biologically, pharmaceutically ortherapeutically active form of the compound.

To produce a prodrug, a pharmaceutically active compound is modifiedsuch that the active compound will be regenerated upon in vivoadministration. The prodrug can be designed to alter the metabolicstability or the transport characteristics of a drug, to mask sideeffects or toxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound. (see, for example, Nogrady (1985) MedicinalChemistry A Biochemical Approach, Oxford University Press, New York,pages 388-392; Silverman (1992), The Organic Chemistry of Drug Designand Drug Action, Academic Press, Inc., San Diego, pages 352-401,Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters,Vol. 4, p. 1985; Rooseboom et al., Pharmacological Reviews, 56:53-102,2004; Miller et al., J. Med. Chem. Vol.46, no. 24, 5097-5116, 2003;Aesop Cho, “Recent Advances in Oral Prodrug Discovery”, Annual Reportsin Medicinal Chemistry, Vol. 41, 395-407, 2006).

Prodrug forms of the herein described compounds, wherein the prodrug ismetabolized in vivo to produce a compound of Formula (I), (II), (IIA) or(IIB) as set forth herein are included within the scope of the claims.In some cases, some of the herein-described compounds may be a prodrugfor another derivative or active compound.

Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. Prodrugs may be designed as reversible drugderivatives, for use as modifiers to enhance drug transport tosite-specific tissues. In some embodiments, the design of a prodrugincreases the effective water solubility. See, e.g., Fedorak et al., Am.J. Physiol., 269:G210-218 (1995); McLoed et al., Gastroenterol,106:405-413 (1994); Hochhaus et al., Biomed. Chrom., 6:283-286 (1992);J. Larsen and H. Bundgaard, Int. J. Pharmaceutics, 37, 87 (1987); J.Larsen et al., Int. J. Pharmaceutics, 47, 103 (1988); Sinkula et al., J.Pharm. Sci., 64:181-210 (1975); T. Higuchi and V. Stella, Pro-drugs asNovel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series; andEdward B. Roche, Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, all incorporatedherein for such disclosure).

Sites on the aromatic ring portion of compounds described herein can besusceptible to various metabolic reactions, therefore incorporation ofappropriate substituents on the aromatic ring structures, such as, byway of example only, halogens can reduce, minimize or eliminate thismetabolic pathway.

The compounds described herein may be labeled isotopically (e.g. with aradioisotope) or by other means, including, but not limited to, the useof chromophores or fluorescent moieties, bioluminescent labels,photoactivatable or chemiluminescent labels.

Compounds described herein include isotopically-labeled compounds, whichare identical to those recited in the various formulae and structurespresented herein, but for the fact that one or more atoms are replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as, forexample, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶C1, respectively.Certain isotopically-labeled compounds described herein, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Further, substitution with isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, such as, for example, increased in vivo half-lifeor reduced dosage requirements.

In additional or further embodiments, the compounds described herein aremetabolized upon administration to an organism in need to produce ametabolite that is then used to produce a desired effect, including adesired therapeutic effect.

Compounds described herein may be formed as, and/or used as,pharmaceutically acceptable salts. The type of pharmaceutical acceptablesalts, include, but are not limited to: (1) acid addition salts, formedby reacting the free base form of the compound with a pharmaceuticallyacceptable: inorganic acid, such as, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid,and the like; or with an organic acid, such as, for example, aceticacid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaricacid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonicacid, toluenesulfonic acid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-l-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, butyric acid, phenylacetic acid,phenylbutyric acid, valproic acid, and the like; (2) salts formed whenan acidic proton present in the parent compound is replaced by a metalion, e.g., an alkali metal ion (e.g. lithium, sodium, potassium), analkaline earth ion (e.g. magnesium, or calcium), or an aluminum ion. Insome cases, compounds described herein may coordinate with an organicbase, such as, but not limited to, ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, dicyclohexylamine,tris(hydroxymethyl)methylamine. In other cases, compounds describedherein may form salts with amino acids such as, but not limited to,arginine, lysine, and the like. Acceptable inorganic bases used to formsalts with compounds that include an acidic proton, include, but are notlimited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide,sodium carbonate, sodium hydroxide, and the like.

It should be understood that a reference to a pharmaceuticallyacceptable salt includes the solvent addition forms or crystal formsthereof, particularly solvates or polymorphs. Solvates contain eitherstoichiometric or non-stoichiometric amounts of a solvent, and may beformed during the process of crystallization with pharmaceuticallyacceptable solvents such as water, ethanol, and the like. Hydrates areformed when the solvent is water, or alcoholates are formed when thesolvent is alcohol. Solvates of compounds described herein can beconveniently prepared or formed during the processes described herein.In addition, the compounds provided herein can exist in unsolvated aswell as solvated forms. In general, the solvated forms are consideredequivalent to the unsolvated forms for the purposes of the compounds andmethods provided herein.

In some embodiments, compounds described herein, such as compounds ofFormula (I), (II), (IIA) and (IIB), are in various forms, including butnot limited to, amorphous forms, milled forms and nano-particulateforms. In addition, compounds described herein include crystallineforms, also known as polymorphs. Polymorphs include the differentcrystal packing arrangements of the same elemental composition of acompound. Polymorphs usually have different X-ray diffraction patterns,melting points, density, hardness, crystal shape, optical properties,stability, and solubility. Various factors such as the recrystallizationsolvent, rate of crystallization, and storage temperature may cause asingle crystal form to dominate.

The screening and characterization of the pharmaceutically acceptablesalts, polymorphs and/or solvates may be accomplished using a variety oftechniques including, but not limited to, thermal analysis, x-raydiffraction, spectroscopy, vapor sorption, and microscopy. Thermalanalysis methods address thermo chemical degradation or thermo physicalprocesses including, but not limited to, polymorphic transitions, andsuch methods are used to analyze the relationships between polymorphicforms, determine weight loss, to find the glass transition temperature,or for excipient compatibility studies. Such methods include, but arenot limited to, Differential scanning calorimetry (DSC), ModulatedDifferential Scanning calorimetry (MDCS), Thermogravimetric analysis(TGA), and Thermogravi-metric and Infrared analysis (TG/IR). X-raydiffraction methods include, but are not limited to, single crystal andpowder diffractometers and synchrotron sources. The variousspectroscopic techniques used include, but are not limited to, Raman,FTIR, UV-VIS, and NMR (liquid and solid state). The various microscopytechniques include, but are not limited to, polarized light microscopy,Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis(EDX), Environmental Scanning Electron Microscopy with EDX (in gas orwater vapor atmosphere), IR microscopy, and Raman microscopy.

Throughout the specification, groups and substituents thereof can bechosen by one skilled in the field to provide stable moieties andcompounds.

Synthesis of Compounds

In some embodiments, the synthesis of compounds described herein areaccomplished using means described in the chemical literature, using themethods described herein, or by a combination thereof. In addition,solvents, temperatures and other reaction conditions presented hereinmay vary according to those of skill in the art.

In other embodiments, the starting materials and reagents used for thesynthesis of the compounds described herein are synthesized or areobtained from commercial sources, such as, but not limited to,Sigma-Aldrich, FischerScientific (Fischer Chemicals), and AcrosOrganics.

In further embodiments, the compounds described herein, and otherrelated compounds having different substituents are synthesized usingtechniques and materials described herein as well as those that areknown in the art, such as described, for example, in Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive OrganicTransformations (VCH Publishers Inc., 1989), March, ADVANCED ORGANICCHEMISTRY 4^(th) Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANICCHEMISTRY 4^(th) Ed., Vols. A and B (Plenum 2000, 2001), and Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3^(rd) Ed., (Wiley 1999)(all of which are incorporated by reference for such disclosure).General methods for the preparation of compound as disclosed herein maybe derived from known reactions in the field, and the reactions may bemodified by the use of appropriate reagents and conditions, as would berecognized by the skilled person, for the introduction of the variousmoieties found in the formulae as provided herein. As a guide thefollowing synthetic methods may be utilized.

Formation of Covalent Linkages by Reaction of an Electrophile with aNucleophile

The compounds described herein can be modified using variouselectrophiles and/or nucleophiles to form new functional groups orsubstituents. Table 2 entitled “Examples of Covalent Linkages andPrecursors Thereof' lists selected non-limiting examples of covalentlinkages and precursor functional groups which yield the covalentlinkages. Table 2 may be used as guidance toward the variety ofelectrophiles and nucleophiles combinations available that providecovalent linkages. Precursor functional groups are shown aselectrophilic groups and nucleophilic groups.

TABLE 2 Examples of Covalent Linkages and Precursors Thereof CovalentLinkage Product Electrophile Nucleophile Carboxamides Activated estersamines/anilines Carboxamides acyl azides amines/anilines Carboxamidesacyl halides amines/anilines Esters acyl halides alcohols/phenols Estersacyl nitriles alcohols/phenols Carboxamides acyl nitrilesamines/anilines Imines Aldehydes amines/anilines Alkyl amines alkylhalides amines/anilines Esters alkyl halides carboxylic acids Thioethersalkyl halides Thiols Ethers alkyl halides alcohols/phenols Thioethersalkyl sulfonates Thiols Esters Anhydrides alcohols/phenols CarboxamidesAnhydrides amines/anilines Thiophenols aryl halides Thiols Aryl aminesaryl halides Amines Thioethers Azindines Thiols Carboxamides carboxylicacids amines/anilines Esters carboxylic acids Alcohols hydrazinesHydrazides carboxylic acids N-acylureas or Anhydrides carbodiimidescarboxylic acids Esters diazoalkanes carboxylic acids ThioethersEpoxides Thiols Thioethers haloacetamides Thiols Ureas Isocyanatesamines/anilines Urethanes Isocyanates alcohols/phenols Thioureasisothiocyanates amines/anilines Thioethers Maleimides Thiols Alkylamines sulfonate esters amines/anilines Thioethers sulfonate estersThiols Sulfonamides sulfonyl halides amines/anilines Sulfonate esterssulfonyl halides phenols/alcohols

Use of Protecting Groups

In the reactions described, it may be necessary to protect reactivefunctional groups, for example hydroxy, amino, imino, thio or carboxygroups, where these are desired in the final product, in order to avoidtheir unwanted participation in reactions. Protecting groups are used toblock some or all of the reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. It is preferred that each protective group be removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval.

Protective groups can be removed by acid, base, reducing conditions(such as, for example, hydrogenolysis), and/or oxidative conditions.Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilylare acid labile and may be used to protect carboxy and hydroxy reactivemoieties in the presence of amino groups protected with Cbz groups,which are removable by hydrogenolysis, and Fmoc groups, which are baselabile. Carboxylic acid and hydroxy reactive moieties may be blockedwith base labile groups such as, but not limited to, methyl, ethyl, andacetyl in the presence of amines blocked with acid labile groups such ast-butyl carbamate or with carbamates that are both acid and base stablebut hydrolytically removable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be protected by conversion to simple ester compounds as exemplifiedherein, which include conversion to alkyl esters, or they may be blockedwith oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups may be blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in then presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with a Pd⁰-catalyzedreaction in the presence of acid labile t-butyl carbamate or base-labileacetate amine protecting groups. Yet another form of protecting group isa resin to which a compound or intermediate may be attached. As long asthe residue is attached to the resin, that functional group is blockedand cannot react. Once released from the resin, the functional group isavailable to react.

Typically blocking/protecting groups may be selected from:

Other protecting groups, plus a detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, N.Y., 1999, and Kocienski,Protective Groups, Thieme Verlag, New York, N.Y., 1994, which areincorporated herein by reference for such disclosure).

General Synthesis

The preparation of compounds of Formula (I), (II), (IIA) and (IIB)described herein may be accomplished by methods known in the art, suchas described in Koebel et al. J. Med. Chem. 1975, vol 18, no 2, 192-194;Gewald, K.; Schinke, E.; Böttcher, H. Chem. Ber. 1966, 99, 94-100;Sabnis, R. W. Sulfur Rep. 1994, 16, 1-17; Sabnis, R. W. et al., J.Heterocyclic Chem. 1999, 36, 333; Gernot A. Eller, Wolfgang HolzerMolecules 2006, 11, 371-376; Michael G. et al., J. Med. Chem.; 1999;42(26) pp 5437-5447; all of which are incorporated by reference.

In one embodiment, compounds described herein are prepared by thesequence depicted in Scheme A.

A Knoevenagel condensation between ketones of structure A-1 andcyanoacetates of structure A-2 forms Schiff's bases of structure A-3.For example ketones of structure A-1 are reacted with cyanoacetates ofstructure A-2 in the presence of an amine, such as for example,morpholine in a solvent such as toluene under dehydrating conditions,such as in the presence of 4A molecular sieves, to form Schiff's base ofstructure A-3. Schiff's base of structure A-3 are reacted under Gewaldreaction conditions (sulfur (S₈), morpholine in a solvent such asethanol and toluene) to form thiophenes of structure A-4. Thiophenes ofstructure A-4 are then reacted with a variety of carboxylic acidchlorides to provide compounds of Formula (I). In another embodiment,thiophenes of structure A-4 can be coupled with carboxylic acids in thepresence of a coupling agent, such as, for example,dicyclohexylcarbodiimide (DCC), diisopropyl carbodiimide (DIC),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI),N-hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (HOSu),4-nitropheno1, pentafluoropheno1,2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), O-benzotriazole-N,N,N′N′-tetramethyluronium hexafluorophosphate(HBTU), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate,bromo-trispyrrolidino-phosphonium hexafluorophosphate,2-(5-norbornene-,3-dicarboximido)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TNTU), O—(N-succinimidyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TSTU), tetramethylfluoroformamidiniumhexafluorophosphate and the like, to provide compounds of Formula (I).

In another embodiment, compounds of Formula (I), (II), (IIA) and (IIB)are prepared by following the procedure outlined in Scheme B.

Ketones of structure A-1, cyanoacetates of structure A-2, elementalsulfur, morpholine, and ethanol are mixed together and stirred at roomtemperature to form thiophenes of structure A-4. Thiophenes of structureA-4 are then reacted with activated carboxylic acids, such as acidchlorides, to form amides of structure A-5. Hydrolysis the esterfunctionality of amides of structure A-5 provides the correspondingcarboxylic acids.

Schemes presented herein are merely illustrative of some methods bywhich the compounds described herein can be synthesized, and variousmodifications to these schemes can be made and will be suggested to oneskilled in the art having referred to this disclosure.

Throughout the specification, groups and substituents thereof can bechosen by one skilled in the field to provide stable moieties andcompounds.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. In the event that thereare a plurality of definitions for terms herein, those in this sectionprevail. All patents, patent applications, publications and publishednucleotide and amino acid sequences (e.g., sequences available inGenBank or other databases) referred to herein are incorporated byreference. Where reference is made to a URL or other such identifier oraddress, it is understood that such identifiers can change andparticular information on the internet can come and go, but equivalentinformation can be found by searching the internet. Reference theretoevidences the availability and public dissemination of such information.

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. In this application,the use of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Definition of standard chemistry terms may be found in reference works,including but not limited to, Carey and Sundberg “ADVANCED ORGANICCHEMISTRY 4^(TH) ED.” Vols. A (2000) and B (2001), Plenum Press, NewYork. Unless otherwise indicated, conventional methods of massspectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinantDNA techniques and pharmacology, within the skill of the art areemployed.

Unless specific definitions are provided, the nomenclature employed inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those known in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients. Standard techniques can be used for recombinantDNA, oligonucleotide synthesis, and tissue culture and transformation(e.g., electroporation, lipofection). Reactions and purificationtechniques can be performed e.g., using kits of manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures can be generallyperformed of conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the methods, compounds, compositions describedherein.

As used herein, C₁-C_(x) includes C₁-C₂, C₁-C₃ . . . C₁-C_(x). C₁-C_(x)refers to the number of carbon atoms that make up the moiety to which itdesignates (excluding optional substituents).

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylgroups may or may not include units of unsaturation. The alkyl moietymay be a “saturated alkyl” group, which means that it does not containany units of unsaturation (i.e. a carbon-carbon double bond or acarbon-carbon triple bond). The alkyl group may also be an “unsaturatedalkyl” moiety, which means that it contains at least one unit ofunsaturation. The alkyl moiety, whether saturated or unsaturated, may bebranched, straight chain, or cyclic.

The “alkyl” group may have 1 to 6 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 6” refers to each integer in thegiven range; e.g., “1 to 6 carbon atoms” means that the alkyl group mayconsist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 6 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). The alkyl group of the compounds described herein may bedesignated as “C₁-C₆ alkyl” or similar designations. By way of exampleonly, “C₁-C₆ alkyl” indicates that there are one to six carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, t-butyl, n-pentyl, iso-pentyl, neo-pentyl, hexyl, propen-3-yl(allyl), cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl. Alkyl groups can be substituted or unsubstituted.Depending on the structure, an alkyl group can be a monoradical or adiradical (i.e., an alkylene group).

An “alkoxy” refers to a “—O-alkyl” group, where alkyl is as definedherein.

The term “alkenyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a double bond that is not part of anaromatic group. That is, an alkenyl group begins with the atoms—C(R)═CR₂, wherein R refers to the remaining portions of the alkenylgroup, which may be the same or different. Non-limiting examples of analkenyl group include —CH═CH₂, —C(CH₃)═CH₂, —CH═CHCH₃, —CH═C(CH₃)₂ and—C(CH₃)═CHCH₃. The alkenyl moiety may be branched, straight chain, orcyclic (in which case, it would also be known as a “cycloalkenyl”group). Alkenyl groups may have 2 to 6 carbons. Alkenyl groups can besubstituted or unsubstituted. Depending on the structure, an alkenylgroup can be a monoradical or a diradical (i.e., an alkenylene group).

The term “alkynyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a triple bond. That is, an alkynylgroup begins with the atoms —C≡C—R, wherein R refers to the remainingportions of the alkynyl group. Non-limiting examples of an alkynyl groupinclude —C≡CH, —C≡CCH₃, —C≡CCH₂CH₃ and —C≡CCH₂CH₂CH₃. The “R” portion ofthe alkynyl moiety may be branched, straight chain, or cyclic. Analkynyl group can have 2 to 6 carbons. Alkynyl groups can be substitutedor unsubstituted. Depending on the structure, an alkynyl group can be amonoradical or a diradical (i.e., an alkynylene group).

“Amino” refers to a —NH₂ group.

The term “alkylamine” or “alkylamino” refers to the —N(alkyl)_(x) _(y)group, where alkyl is as defined herein and x and y are selected fromthe group x=1, y=1 and x=2, y=0. When x=2, the alkyl groups, takentogether with the nitrogen to which they are attached, can optionallyform a cyclic ring system. “Dialkylamino” refers to a —N(alkyl)₂ group,where alkyl is as defined herein.

The term “aromatic” refers to a planar ring having a delocalizedπ-electron system containing 4n+2 π electrons, where n is an integer.Aromatic rings can be formed from five, six, seven, eight, nine, or morethan nine atoms. Aromatics can be optionally substituted. The term“aromatic” includes both aryl groups (e.g., phenyl, naphthalenyl) andheteroaryl groups (e.g., pyridinyl, quinolinyl).

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings can be formedby five, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups can be optionally substituted. Examples of aryl groups include,but are not limited to phenyl, and naphthalenyl. Depending on thestructure, an aryl group can be a monoradical or a diradical (i.e., anarylene group).

“Carboxy” refers to —CO₂H. In some embodiments, carboxy moieties may bereplaced with a “carboxylic acid bioisostere”, which refers to afunctional group or moiety that exhibits similar physical and/orchemical properties as a carboxylic acid moiety. A carboxylic acidbioisostere has similar biological properties to that of a carboxylicacid group. A compound with a carboxylic acid moiety can have thecarboxylic acid moiety exchanged with a carboxylic acid bioisostere andhave similar physical and/or biological properties when compared to thecarboxylic acid-containing compound. For example, in one embodiment, acarboxylic acid bioisostere would ionize at physiological pH to roughlythe same extent as a carboxylic acid group. Examples of bioisoteres of acarboxylic acid include, but are not limited to,

and the like.

The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromaticradical, wherein each of the atoms forming the ring (i.e. skeletalatoms) is a carbon atom. Cycloalkyls may be saturated, or partiallyunsaturated. Cycloalkyls may be fused with an aromatic ring (in whichcase the cycloalkyl is bonded through a non-aromatic ring carbon atom).Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

and the like.

The terms “heteroaryl” or, alternatively, “heteroaromatic” refers to anaryl group that includes one or more ring heteroatoms selected fromnitrogen, oxygen and sulfur. An N-containing “heteroaromatic” or“heteroaryl” moiety refers to an aromatic group in which at least one ofthe skeletal atoms of the ring is a nitrogen atom. Polycyclic heteroarylgroups may be fused or non-fused. Illustrative examples of heteroarylgroups include the following moieties:

and the like.

A “heterocycloalkyl” group or “heteroalicyclic” group refers to acycloalkyl group, wherein at least one skeletal ring atom is aheteroatom selected from nitrogen, oxygen and sulfur. The radicals maybe fused with an aryl or heteroaryl. Illustrative examples ofheterocycloalkyl groups, also referred to as non-aromatic heterocycles,include:

and the like. The term heteroalicyclic also includes all ring forms ofthe carbohydrates, including but not limited to the monosaccharides, thedisaccharides and the oligosaccharides. Unless otherwise noted,heterocycloalkyls have from 2 to 10 carbons in the ring. It isunderstood that when referring to the number of carbon atoms in aheterocycloalkyl, the number of carbon atoms in the heterocycloalkyl isnot the same as the total number of atoms (including the heteroatoms)that make up the heterocycloalkyl (i.e. skeletal atoms of theheterocycloalkyl ring).

The term “halo” or, alternatively, “halogen” means fluoro, chloro, bromoand iodo.

The term “haloalkyl” refers to an alkyl group that is substituted withone or more halogens. The halogens may the same or they may bedifferent. Non-limiting examples of haloalkyls include —CH₂Cl, —CF₃,—CHF₂, —CH₂CF₃, —CF₂CF₃, —CF(CH₃)₃, and the like.

The terms “fluoroalkyl” and “fluoroalkoxy” include alkyl and alkoxygroups, respectively, that are substituted with one or more fluorineatoms. Non-limiting examples of fluoroalkyls include —CF₃, —CHF₂, —CH₂F,—CH₂CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CF(CH₃)₃, and the like. Non-limitingexamples of fluoroalkoxy groups, include —OCF₃, —OCHF₂, —OCH₂F,—OCH₂CF₃, —OCF₂CF₃, —OCF₂CF₂CF₃, —OCF(CH₃)₂, and the like.

The term “heteroalkyl” refers to an alkyl radical where one or moreskeletal chain atoms is selected from an atom other than carbon, e.g.,oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof.The heteroatom(s) may be placed at any interior position of theheteroalkyl group. Examples include, but are not limited to, —CH₂—O—CH₃,—CH₂—CH₂—O—CH₃, —CH₂—NH—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—N(CH₃)—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH₂—NH—OCH₃, —CH₂—O—Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. In addition, up to twoheteroatoms may be consecutive, such as, by way of example, —CH₂—NH—OCH₃and —CH₂—O—Si(CH₃)₃. Excluding the number of heteroatoms, a“heteroalkyl” may have from 1 to 6 carbon atoms.

The term “bond” or “single bond” refers to a chemical bond between twoatoms, or two moieties when the atoms joined by the bond are consideredto be part of larger substructure.

The term “moiety” refers to a specific segment or functional group of amolecule. Chemical moieties are often recognized chemical entitiesembedded in or appended to a molecule.

As used herein, the substituent “R” appearing by itself and without anumber designation refers to a substituent selected from among fromalkyl, haloalkyl, heteroalkyl, alkenyl, cycloalkyl, aryl, heteroaryl(bonded through a ring carbon), and heterocycloalkyl.

The term “optionally substituted” or “substituted” means that thereferenced group may be substituted with one or more additional group(s)individually and independently selected from alkyl, cycloalkyl, aryl,heteroaryl, heterocycloalkyl, —OH, alkoxy, aryloxy, alkylthio, arylthio,alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, —CN, halo,acyl, acyloxy, —CO₂H, —CO₂-alkyl, nitro, haloalkyl, fluoroalkyl, andamino, including mono- and di-substituted amino groups (e.g. —NH₂, —NHR,—N(R)₂), and the protected derivatives thereof. By way of example, anoptional substituents may be LsRs, _(w)h_(ere)i_(n eac)h L^(s) isindependently selected from a bond, —O—, —C(═O)—, —S—, —S(═O)—,—S(═O)₂—, —NH—, —NHC(O)—, —C(O)NH—, S(═O)₂NH—, —NHS(═O)₂, —OC(O)NH—,—NHC(O)O—, —(C₁-C6alkyl)-, or —(C₂-C₆alkenyl)-; and each R^(s) isindependently selected from among H, (C₁-C₆alkyl), (C₃-C₈cycloalkyl),aryl, heteroaryl, heterocycloalkyl, and C₁-C₆heteroalkyl. The protectinggroups that may form the protective derivatives of the abovesubstituents are found in sources such as Greene and Wuts, above.

The methods and formulations described herein include the use ofcrystalline forms (also known as polymorphs), or pharmaceuticallyacceptable salts of compounds having the structure of Formula (I), (II),(IIA) or (IIB), as well as active metabolites of these compounds havingthe same type of activity. In some situations, compounds may exist astautomers. All tautomers are included within the scope of the compoundspresented herein. In addition, the compounds described herein can existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms of thecompounds presented herein are also considered to be disclosed herein.

The terms “kit” and “article of manufacture” are used as synonyms.

The term “subject” or “patient” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of theMammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. In one embodiment of the methods andcompositions provided herein, the mammal is a human.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating a disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying causes of symptoms, inhibiting the disease or condition,e.g., arresting the development of the disease or condition, relievingthe disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

As used herein, the term “target protein” refers to a protein or aportion of a protein capable of being bound by, or interacting with acompound described herein, such as a compound of Formula (I), (II),(IIA) or (IIB). In certain embodiments, a target protein is a STIMprotein. In certain embodiments, a target protein is an Orai protein.

As used herein, “STIM protein” includes but is not limited to, mammalianSTIM-1, such as human and rodent (e.g., mouse) STIM-1, Drosophilamelanogaster D-STIM, C. elegans C-STIM, Anopheles gambiae STIM andmammalian STIM-2, such as human and rodent (e.g., mouse) STIM-2. (seeparagraphs [0211] through [0270] of US 2007/0031814, as well as Table 3of US 2007/0031814, herein incorporated by reference) As describedherein, such proteins have been identified as being involved in,participating in and/or providing for store-operated calcium entry ormodulation thereof, cytoplasmic calcium buffering and/or modulation ofcalcium levels in or movement of calcium into, within or out ofintracellular calcium stores (e.g., endoplasmic reticulum).

As used herein, an “Orai protein” includes Orail (SEQ ID NO: 1 asdescribed in WO 07/081804), Orai2 (SEQ ID NO: 2 as described in WO07/081804), or Orai3 (SEQ ID NO: 3 as described in WO 07/081804). Orailnucleic acid sequence corresponds to GenBank accession number NM 032790,Orai2 nucleic acid sequence corresponds to GenBank accession numberBC069270 and Orai3 nucleic acid sequence corresponds to GenBankaccession number NM_(—)152288. As used herein, Orai refers to any one ofthe Orai genes, e.g., Orai1, Orai2, Orai3 (see Table I of WO 07/081804).As described herein, such proteins have been identified as beinginvolved in, participating in and/or providing for store-operatedcalcium entry or modulation thereof, cytoplasmic calcium bufferingand/or modulation of calcium levels in or movement of calcium into,within or out of intracellular calcium stores (e.g., endoplasmicreticulum).

The term “fragment” or “derivative” when referring to a protein (e.g.STIM, Orai) means proteins or polypeptides which retain essentially thesame biological function or activity in at least one assay as the nativeprotein(s). For example, the fragments or derivatives of the referencedprotein maintains at least about 50% of the activity of the nativeproteins, at least 75%, at least about 95% of the activity of the nativeproteins, as determined e.g. by a calcium influx assay.

As used herein, amelioration of the symptoms of a particular disease,disorder or condition by administration of a particular compound orpharmaceutical composition refers to any lessening of severity, delay inonset, slowing of progression, or shortening of duration, whetherpermanent or temporary, lasting or transient that can be attributed toor associated with administration of the compound or composition.

The term “modulate,” as used herein, means to interact with a targetprotein either directly or indirectly so as to alter the activity of thetarget protein, including, by way of example only, to inhibit theactivity of the target, or to limit or reduce the activity of thetarget.

As used herein, the term “modulator” refers to a compound that alters anactivity of a target. For example, a modulator can cause an increase ordecrease in the magnitude of a certain activity of a target compared tothe magnitude of the activity in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of one or more activities of a target. In certain embodiments,an inhibitor completely prevents one or more activities of a target.

As used herein, “modulation” with reference to intracellular calciumrefers to any alteration or adjustment in intracellular calciumincluding but not limited to alteration of calcium concentration in thecytoplasm and/or intracellular calcium storage organelles, e.g.,endoplasmic reticulum, and alteration of the kinetics of calcium fluxesinto, out of and within cells. In aspect, modulation refers toreduction.

As used herein, the term “target activity” refers to a biologicalactivity capable of being modulated by a modulator. Certain exemplarytarget activities include, but are not limited to, binding affinity,signal transduction, enzymatic activity, tumor growth, inflammation orinflammation-related processes, and amelioration of one or more symptomsassociated with a disease or condition.

The terms “inhibits”, “inhibiting”, or “inhibitor” of SOC channelactivity or CRAC channel activity, as used herein, refer to inhibitionof store operated calcium channel activity or calcium release activatedcalcium channel activity.

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

By “pharmaceutically acceptable,” as used herein, refers a material,such as a carrier or diluent, which does not abrogate the biologicalactivity or properties of the compound, and is relatively nontoxic,i.e., the material may be administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that one activeingredient, e.g. a compound of Formula (I), (II), (IIA) or (IIB), and aco-agent, are both administered to a patient simultaneously in the formof a single entity or dosage. The term “non-fixed combination” meansthat one active ingredient, e.g. a compound of Formula (I), (II), (IIA)or (IIB), and a co-agent, are administered to a patient as separateentities either simultaneously, concurrently or sequentially with nospecific intervening time limits, wherein such administration provideseffective levels of the two compounds in the body of the patient. Thelatter also applies to cocktail therapy, e.g. the administration ofthree or more active ingredients.

The term “pharmaceutical composition” refers to a mixture of a compoundof Formula (I), (II), (IIA) or (IIB) described herein with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to: intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition that includes a compound of Formula (I), (II), (IIA) or(IIB) described herein required to provide a clinically significantdecrease in disease symptoms. An appropriate “effective” amount in anyindividual case may be determined using techniques, such as a doseescalation study.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes) by which a particular substance is changed by anorganism. Thus, enzymes may produce specific structural alterations to acompound. For example, cytochrome P450 catalyzes a variety of oxidativeand reductive reactions while uridine diphosphate glucuronyltransferasescatalyze the transfer of an activated glucuronic-acid molecule toaromatic alcohols, aliphatic alcohols, carboxylic acids, amines and freesulphydryl groups. Further information on metabolism may be obtainedfrom The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill(1996). Metabolites of the compounds disclosed herein can be identifiedeither by administration of compounds to a host and analysis of tissuesamples from the host, or by incubation of compounds with hepatic cellsin vitro and analysis of the resulting compounds. Both methods are wellknown in the art.

“Bioavailability” refers to the percentage of the weight of the compounddisclosed herein (e.g. compound of Formula (I), (II), (IIA) or (IIB)),that is delivered into the general circulation of the animal or humanbeing studied. The total exposure (AUC(0-∞)) of a drug when administeredintravenously is usually defined as 100% bioavailable (F %). “Oralbioavailability” refers to the extent to which a compound disclosedherein, is absorbed into the general circulation when the pharmaceuticalcomposition is taken orally as compared to intravenous injection.

“Blood plasma concentration” refers to the concentration of a compoundof Formula (I), (II), (IIA) or (IIB) disclosed herein, in the plasmacomponent of blood of a subject. It is understood that the plasmaconcentration of compounds described herein may vary significantlybetween subjects, due to variability with respect to metabolism and/orpossible interactions with other therapeutic agents. In accordance withone embodiment disclosed herein, the blood plasma concentration of thecompounds disclosed herein may vary from subject to subject. Likewise,values such as maximum plasma concentration (Cmax) or time to reachmaximum plasma concentration (Tmax), or total area under the plasmaconcentration time curve (AUC(0-∞)) may vary from subject to subject.Due to this variability, the amount necessary to constitute “atherapeutically effective amount” of a compound may vary from subject tosubject.

As used herein, “calcium homeostasis” refers to the maintenance of anoverall balance in intracellular calcium levels and movements, includingcalcium signalling, within a cell.

As used herein, “intracellular calcium” refers to calcium located in acell without specification of a particular cellular location. Incontrast, “cytosolic” or “cytoplasmic” with reference to calcium refersto calcium located in the cell cytoplasm.

As used herein, an effect on intracellular calcium is any alteration ofany aspect of intracellular calcium, including but not limited to, analteration in intracellular calcium levels and location and movement ofcalcium into, out of or within a cell or intracellular calcium store ororganelle. For example, an effect on intracellular calcium can be analteration of the properties, such as, for example, the kinetics,sensitivities, rate, amplitude, and electrophysiologicalcharacteristics, of calcium flux or movement that occurs in a cell orportion thereof An effect on intracellular calcium can be an alterationin any intracellular calcium-modulating process, including,store-operated calcium entry, cytosolic calcium buffering, and calciumlevels in or movement of calcium into, out of or within an intracellularcalcium store. Any of these aspects can be assessed in a variety of waysincluding, but not limited to, evaluation of calcium or other ion(particularly cation) levels, movement of calcium or other ion(particularly cation), fluctuations in calcium or other ion(particularly cation) levels, kinetics of calcium or other ion(particularly cation) fluxes and/or transport of calcium or other ion(particularly cation) through a membrane. An alteration can be any suchchange that is statistically significant. Thus, for example ifintracellular calcium in a test cell and a control cell is said todiffer, such difference can be a statistically significant difference.

As used herein, “involved in” with respect to the relationship between aprotein and an aspect of intracellular calcium or intracellular calciumregulation means that when expression or activity of the protein in acell is reduced, altered or eliminated, there is a concomitant orassociated reduction, alteration or elimination of one or more aspectsof intracellular calcium or intracellular calcium regulation. Such analteration or reduction in expression or activity can occur by virtue ofan alteration of expression of a gene encoding the protein or byaltering the levels of the protein. A protein involved in an aspect ofintracellular calcium, such as, for example, store-operated calciumentry, thus, can be one that provides for or participates in an aspectof intracellular calcium or intracellular calcium regulation. Forexample, a protein that provides for store-operated calcium entry can bea STIM protein and/or an Orai protein.

As used herein, a protein that is a component of a calcium channel is aprotein that participates in multi-protein complex that forms thechannel.

As used herein, “basal” or “resting” with reference to cytosolic calciumlevels refers to the concentration of calcium in the cytoplasm of acell, such as, for example, an unstimulated cell, that has not beensubjected to a condition that results in movement of calcium into or outof the cell or within the cell. The basal or resting cytosolic calciumlevel can be the concentration of free calcium (i.e., calcium that isnot bound to a cellular calcium-binding substance) in the cytoplasm of acell, such as, for example, an unstimulated cell, that has not beensubjected to a condition that results in movement of calcium into or outof the cell.

As used herein, “movement” with respect to ions, including cations,e.g., calcium, refers to movement or relocation, such as for exampleflux, of ions into, out of, or within a cell. Thus, movement of ions canbe, for example, movement of ions from the extracellular medium into acell, from within a cell to the extracellular medium, from within anintracellular organelle or storage site to the cytosol, from the cytosolinto an intracellular organelle or storage site, from one intracellularorganelle or storage site to another intracellular organelle or storagesite, from the extracellular medium into an intracellular organelle orstorage site, from an intracellular organelle or storage site to theextracellular medium and from one location to another within the cellcytoplasm.

As used herein, “cation entry” or “calcium entry” into a cell refers toentry of cations, such as calcium, into an intracellular location, suchas the cytoplasm of a cell or into the lumen of an intracellularorganelle or storage site. Thus, cation entry can be, for example, themovement of cations into the cell cytoplasm from the extracellularmedium or from an intracellular organelle or storage site, or themovement of cations into an intracellular organelle or storage site fromthe cytoplasm or extracellular medium. Movement of calcium into thecytoplasm from an intracellular organelle or storage site is alsoreferred to as “calcium release” from the organelle or storage site.

As used herein, “protein that modulates intracellular calcium” refers toany cellular protein that is involved in regulating, controlling and/oraltering intracellular calcium. For example, such a protein can beinvolved in altering or adjusting intracellular calcium in a number ofways, including, but not limited to, through the maintenance of restingor basal cytoplasmic calcium levels, or through involvement in acellular response to a signal that is transmitted in a cell through amechanism that includes a deviation in intracellular calcium fromresting or basal states. In the context of a “protein that modulatesintracellular calcium,” a “cellular” protein is one that is associatedwith a cell, such as, for example, a cytoplasmic protein, a plasmamembrane-associated protein or an intracellular membrane protein.Proteins that modulate intracellular calcium include, but are notlimited to, ion transport proteins, calcium-binding proteins andregulatory proteins that regulate ion transport proteins.

As used herein, “amelioration” refers to an improvement in a disease orcondition or at least a partial relief of symptoms associated with adisease or condition.

As used herein, “cell response” refers to any cellular response thatresults from ion movement into or out of a cell or within a cell. Thecell response may be associated with any cellular activity that isdependent, at least in part, on ions such as, for example, calcium. Suchactivities may include, for example, cellular activation, geneexpression, endocytosis, exocytosis, cellular trafficking and apoptoticcell death.

As used herein, “immune cells” include cells of the immune system andcells that perform a function or activity in an immune response, suchas, but not limited to, T-cells, B-cells, lymphocytes, macrophages,dendritic cells, neutrophils, eosinophils, basophils, mast cells, plasmacells, white blood cells, antigen presenting cells and natural killercells.

As used herein, “cytokine” refers to small soluble proteins secreted bycells that can alter the behavior or properties of the secreting cell oranother cell. Cytokines bind to cytokine receptors and trigger abehavior or property within the cell, for example, cell proliferation,death or differentiation. Exemplary cytokines include, but are notlimited to, interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18,IL-1α, IL-1β, and IL-1 RA), granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),oncostatin M, erythropoietin, leukemia inhibitory factor (LIF),interferons, B7.1 (also known as CD80), B7.2 (also known as B70, CD86),TNF family members (TNF-α, TNF-β, LT-β, CD40 ligand, Fas ligand, CD27ligand, CD30 ligand, 4-1BBL, Trail), and MIF.

“Store operated calcium entry” or “SOCE” refers to the mechanism bywhich release of calcium ions from intracellular stores is coordinatedwith ion influx across the plasma membrane.

“Selective inhibitor of SOC channel activity” means that the inhibitoris selective for SOC channels and does not substantially affect theactivity of other types of ion channels.

“Selective inhibitor of CRAC channel activity” means that the inhibitoris selective for CRAC channels and does not substantially affect theactivity of other types of ion channels and/or other SOC channels.

Examples of Pharmaceutical Compositions and Methods of Administration

Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art. A summary of pharmaceuticalcompositions described herein may be found, for example, in Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins1999), herein incorporated byreference for such disclosure.

A pharmaceutical composition, as used herein, refers to a mixture of acompound of Formula (I), (II), (IIA) or (IIB) described herein, withother chemical components, such as carriers, stabilizers, diluents,dispersing agents, suspending agents, thickening agents, and/orexcipients. The pharmaceutical composition facilitates administration ofthe compound to an organism. In practicing the methods of treatment oruse provided herein, therapeutically effective amounts of compoundsdescribed herein are administered in a pharmaceutical composition to amammal having a disease, disorder, or condition to be treated. In someembodiments, the mammal is a human. A therapeutically effective amountcan vary widely depending on the severity of the disease, the age andrelative health of the subject, the potency of the compound used andother factors. The compounds of Formula (I), (II), (IIA) or (IIB) can beused singly or in combination with one or more therapeutic agents ascomponents of mixtures (as in combination therapy).

The pharmaceutical formulations described herein can be administered toa subject by multiple administration routes, including but not limitedto, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular),intranasal, buccal, topical, rectal, or transdermal administrationroutes. Moreover, the pharmaceutical compositions described herein,which include a compound of Formula (I), (II), (IIA) or (IIB) describedherein, can be formulated into any suitable dosage form, including butnot limited to, aqueous oral dispersions, liquids, gels, syrups,elixirs, slurries, suspensions, aerosols, controlled releaseformulations, fast melt formulations, effervescent formulations,lyophilized formulations, tablets, powders, pills, dragees, capsules,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations, and mixed immediaterelease and controlled release formulations.

One may administer the compounds and/or compositions in a local ratherthan systemic manner, for example, via injection of the compounddirectly into an organ or tissue, often in a depot preparation orsustained release formulation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Furthermore, one mayadminister the drug in a targeted drug delivery system, for example, ina liposome coated with organ-specific antibody. The liposomes will betargeted to and taken up selectively by the organ. In addition, the drugmay be provided in the form of a rapid release formulation, in the formof an extended release formulation, or in the form of an intermediaterelease formulation.

Pharmaceutical compositions including a compound described herein may bemanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The pharmaceutical compositions will include at least one compound ofFormula (I), (II), (IIA) or (IIB) described herein, as an activeingredient in free-acid or free-base form, or in a pharmaceuticallyacceptable salt form. In addition, the methods and pharmaceuticalcompositions described herein include the use of crystalline forms (alsoknown as polymorphs), as well as active metabolites of these compoundshaving the same type of activity. In some situations, compounds mayexist as tautomers. All tautomers are included within the scope of thecompounds presented herein. Additionally, the compounds described hereincan exist in unsolvated as well as solvated forms with pharmaceuticallyacceptable solvents such as water, ethanol, and the like. The solvatedforms of the compounds presented herein are also considered to bedisclosed herein.

In certain embodiments, compositions provided herein may also includeone or more preservatives to inhibit microbial activity. Suitablepreservatives include quaternary ammonium compounds such as benzalkoniumchloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the compounds describedherein (e.g. compounds of Formula (I), (II), (IIA) or (IIB)), optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets, pills,or capsules. Suitable excipients include, for example, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methylcellulose,microcrystalline cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP orpovidone) or calcium phosphate. If desired, disintegrating agents may beadded, such as the cross-linked croscarmellose sodium,polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

In some embodiments, the solid dosage forms disclosed herein may be inthe form of a tablet, (including a suspension tablet, a fast-melttablet, a bite-disintegration tablet, a rapid-disintegration tablet, aneffervescent tablet, or a caplet), a pill, a powder (including a sterilepackaged powder, a dispensable powder, or an effervescent powder), acapsule (including both soft or hard capsules, e.g., capsules made fromanimal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”),solid dispersion, solid solution, bioerodible dosage form, controlledrelease formulations, pulsatile release dosage forms, multiparticulatedosage forms, pellets, granules, or an aerosol. In other embodiments,the pharmaceutical formulation is in the form of a powder. In stillother embodiments, the pharmaceutical formulation is in the form of atablet, including but not limited to, a fast-melt tablet. Additionally,pharmaceutical formulations of the compounds described herein may beadministered as a single capsule or in multiple capsule dosage form. Insome embodiments, the pharmaceutical formulation is administered in two,or three, or four, capsules or tablets.

In some embodiments, solid dosage forms, e.g., tablets, effervescenttablets, and capsules, are prepared by mixing particles of a compound ofFormula (I), (II), (IIA) or (IIB) described herein, with one or morepharmaceutical excipients to form a bulk blend composition. Whenreferring to these bulk blend compositions as homogeneous, it is meantthat the particles of the compound of Formula (I), (II), (IIA) or (IIB)described herein, are dispersed evenly throughout the composition sothat the composition may be readily subdivided into equally effectiveunit dosage forms, such as tablets, pills, and capsules. The individualunit dosages may also include film coatings, which disintegrate uponoral ingestion or upon contact with diluent. These formulations can bemanufactured by conventional pharmacological techniques.

The pharmaceutical solid dosage forms described herein can include acompound of Formula (I), (II), (IIA) or (IIB) described herein, and oneor more pharmaceutically acceptable additives such as a compatiblecarrier, binder, filling agent, suspending agent, flavoring agent,sweetening agent, disintegrating agent, dispersing agent, surfactant,lubricant, colorant, diluent, solubilizer, moistening agent,plasticizer, stabilizer, penetration enhancer, wetting agent,anti-foaming agent, antioxidant, preservative, or one or morecombination thereof. In still other aspects, using standard coatingprocedures, such as those described in Remington's PharmaceuticalSciences, 20th Edition (2000), a film coating is provided around theformulation of the compound described herein. In one embodiment, some orall of the particles of the compound described herein are coated. Inanother embodiment, some or all of the particles of the compounddescribed herein are microencapsulated. In still another embodiment, theparticles of the compound described herein are not microencapsulated andare uncoated.

Suitable carriers for use in the solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like.

Suitable filling agents for use in the solid dosage forms describedherein include, but are not limited to, lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, hydroxypropylmethycellulose(HPMC), hydroxypropylmethycellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose,xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethyleneglycol, and the like.

In order to release the compound of Formula (I), (II), (IIA) or (IIB)from a solid dosage form matrix as efficiently as possible,disintegrants are often used in the formulation, especially when thedosage forms are compressed with binder. Disintegrants help rupturingthe dosage form matrix by swelling or capillary action when moisture isabsorbed into the dosage form. Suitable disintegrants for use in thesolid dosage forms described herein include, but are not limited to,natural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolkaFloc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that canbe filled into soft or hard shell capsules and for tablet formulation,they ensure the tablet remaining intact after compression and helpassure blend uniformity prior to a compression or fill step. Materialssuitable for use as binders in the solid dosage forms described hereininclude, but are not limited to, carboxymethylcellulose, methylcellulose(e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USPPharmacoat-603, hydroxypropylmethylcellulose acetate stearate (AqoateHS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g.,Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystallinecellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesiumaluminum silicate, polysaccharide acids, bentonites, gelatin,polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone,starch, pregelatinized starch, tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such asacacia, tragacanth, ghatti gum, mucilage of isapol husks, starch,polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone®XL-10, and Povidone® K-12), larch arabogalactan, Veegum®, polyethyleneglycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself can act as moderatebinder. Formulators skilled in art can determine the binder level forthe formulations, but binder usage level of up to 70% in tabletformulations is common.

Suitable lubricants or glidants for use in the solid dosage formsdescribed herein include, but are not limited to, stearic acid, calciumhydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal andalkaline earth metal salts, such as aluminum, calcium, magnesium, zinc,stearic acid, sodium stearates, magnesium stearate, zinc stearate,waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol or a methoxypolyethylene glycolsuch as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol,sodium oleate, glyceryl behenate, glyceryl palmitostearate, glycerylbenzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described hereininclude, but are not limited to, sugars (including lactose, sucrose, anddextrose), polysaccharides (including dextrates and maltodextrin),polyols (including mannitol, xylitol, and sorbitol), cyclodextrins andthe like.

Suitable wetting agents for use in the solid dosage forms describedherein include, for example, oleic acid, glyceryl monostearate, sorbitanmonooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodiumoleate, sodium lauryl sulfate, magnesium stearate, sodium docusate,triacetin, vitamin E TPGS and the like.

Suitable surfactants for use in the solid dosage forms described hereininclude, for example, sodium lauryl sulfate, sorbitan monooleate,polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bilesalts, glyceryl monostearate, copolymers of ethylene oxide and propyleneoxide, e.g., Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms describedhere include, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 5400 to about 7000, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Suitable antioxidants for use in the solid dosage forms described hereininclude, for example, e.g., butylated hydroxytoluene (BHT), sodiumascorbate, and tocopherol.

It should be appreciated that there is considerable overlap betweenadditives used in the solid dosage forms described herein. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included in solid dosageforms of the pharmaceutical compositions described herein. The amountsof such additives can be readily determined by one skilled in the art,according to the particular properties desired.

In other embodiments, one or more layers of the pharmaceuticalformulation are plasticized. Illustratively, a plasticizer is generallya high boiling point solid or liquid. Suitable plasticizers can be addedfrom about 0.01% to about 50% by weight (w/w) of the coatingcomposition. Plasticizers include, but are not limited to, diethylphthalate, citrate esters, polyethylene glycol, glycerol, acetylatedglycerides, triacetin, polypropylene glycol, polyethylene glycol,triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, andcastor oil.

Compressed tablets are solid dosage forms prepared by compacting thebulk blend of the formulations described above. In various embodiments,compressed tablets which are designed to dissolve in the mouth willinclude one or more flavoring agents. In other embodiments, thecompressed tablets will include a film surrounding the final compressedtablet. In some embodiments, the film coating can provide a delayedrelease of the compounds of Formula (I), (II), (IIA) or (IIB) describedherein from the formulation. In other embodiments, the film coating aidsin patient compliance (e.g., Opadry® coatings or sugar coating). Filmcoatings including Opadry® typically range from about 1% to about 3% ofthe tablet weight. In other embodiments, the compressed tablets includeone or more excipients.

A capsule may be prepared, for example, by placing the bulk blend of theformulation of the compound described above, inside of a capsule. Insome embodiments, the formulations (non-aqueous suspensions andsolutions) are placed in a soft gelatin capsule. In other embodiments,the formulations are placed in standard gelatin capsules or non-gelatincapsules such as capsules comprising HPMC. In other embodiments, theformulation is placed in a sprinkle capsule, wherein the capsule may beswallowed whole or the capsule may be opened and the contents sprinkledon food prior to eating. In some embodiments, the therapeutic dose issplit into multiple (e.g., two, three, or four) capsules. In someembodiments, the entire dose of the formulation is delivered in acapsule form.

In various embodiments, the particles of the compound of Formula (I),(II), (IIA) or (IIB) described herein and one or more excipients are dryblended and compressed into a mass, such as a tablet, having a hardnesssufficient to provide a pharmaceutical composition that substantiallydisintegrates within less than about 30 minutes, less than about 35minutes, less than about 40 minutes, less than about 45 minutes, lessthan about 50 minutes, less than about 55 minutes, or less than about 60minutes, after oral administration, thereby releasing the formulationinto the gastrointestinal fluid.

In another aspect, dosage forms may include microencapsulatedformulations. In some embodiments, one or more other compatiblematerials are present in the microencapsulation material. Exemplarymaterials include, but are not limited to, pH modifiers, erosionfacilitators, anti-foaming agents, antioxidants, flavoring agents, andcarrier materials such as binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, and diluents.

Materials useful for the microencapsulation described herein includematerials compatible with compounds described herein, which sufficientlyisolate the compound from other non-compatible excipients. Materialscompatible with compounds of Formula (I), (II), (IIA) or (IIB) describedherein are those that delay the release of the compounds of Formula (I),(II), (IIA) or (IIB) in vivo.

Exemplary microencapsulation materials useful for delaying the releaseof the formulations including compounds described herein, include, butare not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel®or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC),hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, BenecelMP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A,hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS)and Metolose®, Ethylcelluloses (EC) and mixtures thereof such as E461,Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such asOpadry AMB, hydroxyethylcelluloses such as Natrosol®,carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) suchas Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymerssuch as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX),polyethylene glycols, modified food starch, acrylic polymers andmixtures of acrylic polymers with cellulose ethers such as Eudragit®EPO, Eudragit® L30D-55, Eudragit® FS 30D Eudragit® L100-55, Eudragit®L100, Eudragit® S100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5,Eudragit® S12.5, Eudragit® NE30D, and Eudragit® NE 40D, celluloseacetate phthalate, sepifilms such as mixtures of HPMC and stearic acid,cyclodextrins, and mixtures of these materials.

In still other embodiments, plasticizers such as polyethylene glycols,e.g., PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,stearic acid, propylene glycol, oleic acid, and triacetin areincorporated into the microencapsulation material. In other embodiments,the microencapsulating material useful for delaying the release of thepharmaceutical compositions is from the USP or the National Formulary(NF). In yet other embodiments, the microencapsulation material isKlucel. In still other embodiments, the microencapsulation material ismethocel.

Microencapsulated compounds of Formula (I), (II), (IIA) or (IIB)described herein may be formulated by methods known by one of ordinaryskill in the art. Such known methods include, e.g., spray dryingprocesses, spinning disk-solvent processes, hot melt processes, spraychilling methods, fluidized bed, electrostatic deposition, centrifugalextrusion, rotational suspension separation, polymerization atliquid-gas or solid-gas interface, pressure extrusion, or sprayingsolvent extraction bath. In addition to these, several chemicaltechniques, e.g., complex coacervation, solvent evaporation,polymer-polymer incompatibility, interfacial polymerization in liquidmedia, in situ polymerization, in-liquid drying, and desolvation inliquid media could also be used. Furthermore, other methods such asroller compaction, extrusion/spheronization, coacervation, ornanoparticle coating may also be used.

In still other embodiments, effervescent powders are also prepared inaccordance with the present disclosure. Effervescent salts have beenused to disperse medicines in water for oral administration.Effervescent salts are granules or coarse powders containing a medicinalagent in a dry mixture, usually composed of sodium bicarbonate, citricacid and/or tartaric acid. When such salts are added to water, the acidsand the base react to liberate carbon dioxide gas, thereby causing“effervescence.” Examples of effervescent salts include, e.g., thefollowing ingredients: sodium bicarbonate or a mixture of sodiumbicarbonate and sodium carbonate, citric acid and/or tartaric acid. Anyacid-base combination that results in the liberation of carbon dioxidecan be used in place of the combination of sodium bicarbonate and citricand tartaric acids, as long as the ingredients were suitable forpharmaceutical use and result in a pH of about 6.0 or higher.

In other embodiments, the formulations described herein, which include acompound described herein, are solid dispersions. Methods of producingsuch solid dispersions are known in the art and include, but are notlimited to, for example, U.S. Pat. Nos. 4,343,789, 5,340,591, 5,456,923,5,700,485, 5,723,269, and U.S. patent publication no. 2004/0013734. Instill other embodiments, the formulations described herein are solidsolutions. Solid solutions incorporate a substance together with theactive agent and other excipients such that heating the mixture resultsin dissolution of the drug and the resulting composition is then cooledto provide a solid blend which can be further formulated or directlyadded to a capsule or compressed into a tablet. Methods of producingsuch solid solutions are known in the art and include, but are notlimited to, for example, U.S. Pat. Nos. 4,151,273, 5,281,420, and6,083,518.

The pharmaceutical solid oral dosage forms including formulationsdescribed herein, which include a compound of Formula (I), (II), (IIA)or (IIB) described herein, can be further formulated to provide acontrolled release of the compound of Formula (I), (II), (IIA) or (IIB).Controlled release refers to the release of the compound of Formula (I),(II), (IIA) or (IIB) described herein from a dosage form in which it isincorporated according to a desired profile over an extended period oftime. Controlled release profiles include, for example, sustainedrelease, prolonged release, pulsatile release, and delayed releaseprofiles. In contrast to immediate release compositions, controlledrelease compositions allow delivery of an agent to a subject over anextended period of time according to a predetermined profile. Suchrelease rates can provide therapeutically effective levels of agent foran extended period of time and thereby provide a longer period ofpharmacologic response while minimizing side effects as compared toconventional rapid release dosage forms. Such longer periods of responseprovide for many inherent benefits that are not achieved with thecorresponding short acting, immediate release preparations.

In some embodiments, the solid dosage forms described herein can beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the smallintestine of the gastrointestinal tract. The enteric coated dosage formmay be a compressed or molded or extruded tablet/mold (coated oruncoated) containing granules, powder, pellets, beads or particles ofthe active ingredient and/or other composition components, which arethemselves coated or uncoated. The enteric coated oral dosage form mayalso be a capsule (coated or uncoated) containing pellets, beads orgranules of the solid carrier or the composition, which are themselvescoated or uncoated.

The term “delayed release” as used herein refers to the delivery so thatthe release can be accomplished at some generally predictable locationin the intestinal tract more distal to that which would have beenaccomplished if there had been no delayed release alterations. In someembodiments the method for delay of release is coating. Any coatingsshould be applied to a sufficient thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below about 5,but does dissolve at pH about 5 and above. Coatings may be made from:

Acrylic polymers. The performance of acrylic polymers (primarily theirsolubility in biological fluids) can vary based on the degree and typeof substitution. Examples of suitable acrylic polymers includemethacrylic acid copolymers and ammonium methacrylate copolymers. TheEudragit series E, L, S, RL, RS and NE (Rohm Pharma) are available assolubilized in organic solvent, aqueous dispersion, or dry powders. TheEudragit series RL, NE, and RS are insoluble in the gastrointestinaltract but are permeable and are used primarily for colonic targeting.The Eudragit series E dissolve in the stomach. The Eudragit series L,L-30D and S are insoluble in stomach and dissolve in the intestine;

Cellulose Derivatives. Examples of suitable cellulose derivatives are:ethyl cellulose; reaction mixtures of partial acetate esters ofcellulose with phthalic anhydride. The performance can vary based on thedegree and type of substitution. Cellulose acetate phthalate (CAP)dissolves in pH>6. Aquateric (FMC) is an aqueous based system and is aspray dried CAP psuedolatex with particles <1 μm. Other components inAquateric can include pluronics, Tweens, and acetylated monoglycerides.Other suitable cellulose derivatives include: cellulose acetatetrimellitate (Eastman); methylcellulose (Pharmacoat, Methocel);hydroxypropylmethyl cellulose phthalate (HPMCP); hydroxypropylmethylcellulose succinate (HPMCS); and hydroxypropylmethylcellulose acetatesuccinate (e.g., AQOAT (Shin Etsu)). The performance can vary based onthe degree and type of substitution. For example, HPMCP such as, HP-50,HP-55, HP-555, HP-55F grades are suitable. The performance can varybased on the degree and type of substitution. For example, suitablegrades of hydroxypropylmethylcellulose acetate succinate include, butare not limited to, AS-LG (LF), which dissolves at pH 5, AS-MG (MF),which dissolves at pH 5.5, and AS-HG (HF), which dissolves at higher pH.These polymers are offered as granules, or as fine powders for aqueousdispersions;

Poly Vinyl Acetate Phthalate (PVAP). PVAP dissolves in pH >5, and it ismuch less permeable to water vapor and gastric fluids.

In some embodiments, the coating can, and usually does, contain aplasticizer and possibly other coating excipients such as colorants,talc, and/or magnesium stearate, which are well known in the art.Suitable plasticizers include triethyl citrate (Citroflex 2), triacetin(glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, anionic carboxylic acrylicpolymers usually will contain 10-25% by weight of a plasticizer,especially dibutyl phthalate, polyethylene glycol, triethyl citrate andtriacetin. Conventional coating techniques such as spray or pan coatingare employed to apply coatings. The coating thickness must be sufficientto ensure that the oral dosage form remains intact until the desiredsite of topical delivery in the intestinal tract is reached.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants(e.g., carnuba wax or PEG) may be added to the coatings besidesplasticizers to solubilize or disperse the coating material, and toimprove coating performance and the coated product.

In other embodiments, the formulations described herein, which include acompound of Formula (I), (II), (IIA) or (IIB) described herein, aredelivered using a pulsatile dosage form. A pulsatile dosage form iscapable of providing one or more immediate release pulses atpredetermined time points after a controlled lag time or at specificsites. Pulsatile dosage forms may be administered using a variety ofpulsatile formulations known in the art. For example, such formulationsinclude, but are not limited to, those described in U.S. Pat. Nos.5,011,692; 5,017,381; 5,229,135; 5,840,329; 4,871,549; 5,260,068;5,260,069; 5,508,040; 5,567,441 and 5,837,284.

Many other types of controlled release systems are suitable for use withthe formulations described herein. Examples of such delivery systemsinclude, e.g., polymer-based systems, such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; porous matrices,nonpolymer-based systems that are lipids, including sterols, such ascholesterol, cholesterol esters and fatty acids, or neutral fats, suchas mono-, di- and triglycerides; hydrogel release systems; silasticsystems; peptide-based systems; wax coatings, bioerodible dosage forms,compressed tablets using conventional binders and the like. See, e.g.,Liberman et al., Pharmaceutical Dosage Forms, 2 Ed., Vol. 1, pp. 209-214(1990); Singh et al., Encyclopedia of Pharmaceutical Technology, 2ndEd., pp. 751-753 (2002); U.S. Pat. Nos. 4,327,725; 4,624,848; 4,968,509;5,461,140; 5,456,923; 5,516,527; 5,622,721; 5,686,105; 5,700,410;5,977,175; 6,465,014; and 6,932,983.

In some embodiments, pharmaceutical formulations are provided thatinclude particles of the compounds described herein, e.g. compounds ofFormula (I), (II), (IIA) or (IIB), and at least one dispersing agent orsuspending agent for oral administration to a subject. The formulationsmay be a powder and/or granules for suspension, and upon admixture withwater, a substantially uniform suspension is obtained.

Liquid formulation dosage forms for oral administration can be aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regard to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another embodiment, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

The pharmaceutical compositions described herein may include sweeteningagents such as, but not limited to, acacia syrup, acesulfame K, alitame,anise, apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate(MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mintcream, mixed berry, neohesperidine DC, neotame, orange, pear, peach,peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer,rum, saccharin, safrole, sorbitol, spearmint, spearmint cream,strawberry, strawberry cream, stevia, sucralose, sucrose, sodiumsaccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin,sucralose, sorbitol, swiss cream, tagatose, tangerine, thaumatin, tuttifruitti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol,or any combination of these flavoring ingredients, e.g., anise-menthol,cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint,honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream,vanilla-mint, and mixtures thereof.

In some embodiments, the pharmaceutical formulations described hereincan be self-emulsifying drug delivery systems (SEDDS). Emulsions aredispersions of one immiscible phase in another, usually in the form ofdroplets. Generally, emulsions are created by vigorous mechanicaldispersion. SEDDS, as opposed to emulsions or microemulsions,spontaneously form emulsions when added to an excess of water withoutany external mechanical dispersion or agitation. An advantage of SEDDSis that only gentle mixing is required to distribute the dropletsthroughout the solution. Additionally, water or the aqueous phase can beadded just prior to administration, which ensures stability of anunstable or hydrophobic active ingredient. Thus, the SEDDS provides aneffective delivery system for oral and parenteral delivery ofhydrophobic active ingredients. SEDDS may provide improvements in thebioavailability of hydrophobic active ingredients. Methods of producingself-emulsifying dosage forms are known in the art and include, but arenot limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and6,960,563.

It is to be appreciated that there is overlap between the above-listedadditives used in the aqueous dispersions or suspensions describedherein, since a given additive is often classified differently bydifferent practitioners in the field, or is commonly used for any ofseveral different functions. Thus, the above-listed additives should betaken as merely exemplary, and not limiting, of the types of additivesthat can be included in formulations described herein. The amounts ofsuch additives can be readily determined by one skilled in the art,according to the particular properties desired.

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452. Formulationsthat include a compound described herein, which are prepared accordingto these and other techniques well-known in the art are prepared assolutions in saline, employing benzyl alcohol or other suitablepreservatives, fluorocarbons, and/or other solubilizing or dispersingagents known in the art. See, for example, Ansel, H. C. et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995).Preferably these compositions and formulations are prepared withsuitable nontoxic pharmaceutically acceptable ingredients. Theseingredients are known to those skilled in the preparation of nasaldosage forms and some of these can be found in REMINGTON: THE SCIENCEAND PRACTICE OF PHARMACY, 21st edition, 2005, a standard reference inthe field. The choice of suitable carriers is highly dependent upon theexact nature of the nasal dosage form desired, e.g., solutions,suspensions, ointments, or gels. Nasal dosage forms generally containlarge amounts of water in addition to the active ingredient. Minoramounts of other ingredients such as pH adjusters, emulsifiers ordispersing agents, preservatives, surfactants, gelling agents, orbuffering and other stabilizing and solubilizing agents may also bepresent. Preferably, the nasal dosage form should be isotonic with nasalsecretions.

For administration by inhalation, the compounds described herein may bein a form as an aerosol, a mist or a powder. Pharmaceutical compositionsdescribed herein are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound described herein and a suitable powder base such aslactose or starch.

Buccal formulations that include compounds described herein may beadministered using a variety of formulations known in the art. Forexample, such formulations include, but are not limited to, U.S. Pat.Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136. In addition, thebuccal dosage forms described herein can further include a bioerodible(hydrolysable) polymeric carrier that also serves to adhere the dosageform to the buccal mucosa. The buccal dosage form is fabricated so as toerode gradually over a predetermined time period, wherein the deliveryof the compound is provided essentially throughout. Buccal drugdelivery, as will be appreciated by those skilled in the art, avoids thedisadvantages encountered with oral drug administration, e.g., slowabsorption, degradation of the active agent by fluids present in thegastrointestinal tract and/or first-pass inactivation in the liver. Withregard to the bioerodible (hydrolysable) polymeric carrier, it will beappreciated that virtually any such carrier can be used, so long as thedesired drug release profile is not compromised, and the carrier iscompatible with the compound of Formula (I), (II), (IIA) or (IIB)described herein, and any other components that may be present in thebuccal dosage unit. Generally, the polymeric carrier compriseshydrophilic (water-soluble and water-swellable) polymers that adhere tothe wet surface of the buccal mucosa. Examples of polymeric carriersuseful herein include acrylic acid polymers and co, e.g., those known as“carbomers” (Carbopol®, which may be obtained from B.F. Goodrich, is onesuch polymer). Other components may also be incorporated into the buccaldosage forms described herein include, but are not limited to,disintegrants, diluents, binders, lubricants, flavoring, colorants,preservatives, and the like. For buccal or sublingual administration,the compositions may take the form of tablets, lozenges, or gelsformulated in a conventional manner.

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiment, the transdermal formulations described hereininclude at least three components: (1) a formulation of a compound ofFormula (I), (II), (IIA) or (IIB); (2) a penetration enhancer; and (3)an aqueous adjuvant. In addition, transdermal formulations can includeadditional components such as, but not limited to, gelling agents,creams and ointment bases, and the like. In some embodiments, thetransdermal formulation can further include a woven or non-woven backingmaterial to enhance absorption and prevent the removal of thetransdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein can maintain a saturated orsupersaturated state to promote diffusion into the skin.

Formulations suitable for transdermal administration of compoundsdescribed herein may employ transdermal delivery devices and transdermaldelivery patches and can be lipophilic emulsions or buffered, aqueoussolutions, dissolved and/or dispersed in a polymer or an adhesive. Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents. Still further, transdermal deliveryof the compounds described herein can be accomplished by means ofiontophoretic patches and the like. Additionally, transdermal patchescan provide controlled delivery of the compound of Formula (I), (II),(IIA) or (IIB) described herein. The rate of absorption can be slowed byusing rate-controlling membranes or by trapping the compound within apolymer matrix or gel. Conversely, absorption enhancers can be used toincrease absorption. An absorption enhancer or carrier can includeabsorbable pharmaceutically acceptable solvents to assist passagethrough the skin. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound to the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

Formulations suitable for intramuscular, subcutaneous, or intravenousinjection may include physiologically acceptable sterile aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection may also contain additives such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the growth ofmicroorganisms can be ensured by various antibacterial and antifungalagents, such as parabens, chlorobutanol, phenol, sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, compounds described herein may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For other parenteral injections, appropriateformulations may include aqueous or nonaqueous solutions, preferablywith physiologically compatible buffers or excipients. Such excipientsare generally known in the art.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

In certain embodiments, delivery systems for pharmaceutical compoundsmay be employed, such as, for example, liposomes and emulsions. Incertain embodiments, compositions provided herein can also include anmucoadhesive polymer, selected from among, for example,carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

In some embodiments, the compounds described herein may be administeredtopically and can be formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. Suchpharmaceutical compounds can contain solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives.

The compounds described herein may also be formulated in rectalcompositions such as enemas, rectal gels, rectal foams, rectal aerosols,suppositories, jelly suppositories, or retention enemas, containingconventional suppository bases such as cocoa butter or other glycerides,as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and thelike. In suppository forms of the compositions, a low-melting wax suchas, but not limited to, a mixture of fatty acid glycerides, optionallyin combination with cocoa butter is first melted.

Generally, an agent, such as a compound of Formula (I), (II), (IIA) or(IIB), is administered in an amount effective for amelioration of, orprevention of the development of symptoms of, the disease or disorder(i.e., a therapeutically effective amount). Thus, a therapeuticallyeffective amount can be an amount that is capable of at least partiallypreventing or reversing a disease or disorder. The dose required toobtain an effective amount may vary depending on the agent, formulation,disease or disorder, and individual to whom the agent is administered.

Determination of effective amounts may also involve in vitro assays inwhich varying doses of agent are administered to cells in culture andthe concentration of agent effective for ameliorating some or allsymptoms is determined in order to calculate the concentration requiredin vivo. Effective amounts may also be based in in vivo animal studies.

An agent can be administered prior to, concurrently with and subsequentto the appearance of symptoms of a disease or disorder. In someembodiments, an agent is administered to a subject with a family historyof the disease or disorder, or who has a phenotype that may indicate apredisposition to a disease or disorder, or who has a genotype whichpredisposes the subject to the disease or disorder.

The particular delivery system used can depend on a number of factors,including, for example, the intended target and the route ofadministration, e.g., local or systemic. Targets for delivery can bespecific cells which are causing or contributing to a disease ordisorder, including, for example, cells that have altered intracellularcalcium or calcium dysregulation or dyshomeostasis, and cells that donot have altered intracellular calcium but that may have somealteration, defect or deficiency that can be, at least in part,compensated, counteracted, reversed or alleviated or eliminated byaltering intracellular calcium of the cell. Particular cells include,for example, immune cells (e.g., lymphocytes, T cells, B cells, whiteblood cells), fibroblasts (or cells derived from a fibroblast),epidermal, dermal or skin cells (e.g., a keratinocytes), blood cells,kidney or renal cells (e.g., mesangial cells), muscle cells (e.g., asmooth muscle cell such as an airway (tracheal or bronchial) smoothmuscle cell) and exocrine or secretory (e.g., salivary, includingparotid acinar and submandibular gland) cells. For example, a targetcell can be resident or infiltrating cells in the lungs or airways thatcontribute to an asthmatic illness or disease, resident or infiltratingcells in the nervous system contributing to a neurological,neurodegenerative or demyelinating disease or disorder, resident orinfiltrating cells involved in rejection of a kidney graft, graftedcells that when activated lead to graft-versus-host disease, resident orinfiltrating cells involved in rejection of a kidney graft, resident orinfiltrating cells, activation of which contributes to inflammation,e.g., in arthritis, resident or infiltrating cells in the kidney orrenal system (e.g., mesangial cells) involved in neuropathy andglomerulonephritis and resident or infiltrating cells in exocrine glands(e.g., salivary and lacrimal glands) involved in autoimmune disorders(e.g., Sjogren's disease). Administration of an agent can be directed toone or more cell types or subsets of a cell type by methods known tothose of skill in the art. For example, an agent can be coupled to anantibody, ligand to a cell surface receptor or a toxin, or can becontained in a particle that is selectively internalized into cells,e.g., liposomes or a virus in which the viral receptor bindsspecifically to a certain cell type, or a viral particle lacking theviral nucleic acid, or can be administered locally.

Examples of Methods of Dosing and Treatment Regimens

The compounds described herein can be used in the preparation ofmedicaments for the modulation of intracellular calcium, or for thetreatment of diseases or conditions that would benefit, at least inpart, from modulation of intracellular calcium. In addition, a methodfor treating any of the diseases or conditions described herein in asubject in need of such treatment, involves administration ofpharmaceutical compositions containing at least one compound describedherein, or a pharmaceutically acceptable salt, pharmaceuticallyacceptable prodrug, or pharmaceutically acceptable solvate thereof, intherapeutically effective amounts to said subject.

The compositions containing the compound(s) described herein can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the compositions are administered to a patientalready suffering from a disease or condition, in an amount sufficientto cure or at least partially arrest the symptoms of the disease orcondition. Amounts effective for this use will depend on the severityand course of the disease or condition, previous therapy, the patient'shealth status, weight, and response to the drugs, and the judgment ofthe treating physician.

In prophylactic applications, compositions containing the compoundsdescribed herein are administered to a patient susceptible to orotherwise at risk of a particular disease, disorder or condition. Suchan amount is defined to be a “prophylactically effective amount ordose.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. When used in a patient, effectiveamounts for this use will depend on the severity and course of thedisease, disorder or condition, previous therapy, the patient's healthstatus and response to the drugs, and the judgment of the treatingphysician.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the compounds may beadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisease or condition.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from about 10%to about 100%, including, by way of example only, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or about 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained. Patients can, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, disease orcondition and its severity, the identity (e.g., weight) of the subjector host in need of treatment, but can nevertheless be determined in amanner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, the condition being treated,and the subject or host being treated. In general, however, dosesemployed for adult human treatment will typically be in the range ofabout 0.02- about 5000 mg per day, in some embodiments, about 1- about1500 mg per day. The desired dose may conveniently be presented in asingle dose or as divided doses administered simultaneously (or over ashort period of time) or at appropriate intervals, for example as two,three, four or more sub-doses per day.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more compound. The unit dosage may bein the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection may be presented in unitdosage form, which include, but are not limited to ampoules, or inmulti-dose containers, with an added preservative.

The daily dosages appropriate for the compounds described hereindescribed herein are from about 0.01 mg/kg to about 20 mg/kg. In oneembodiment, the daily dosages are from about 0.1 mg/kg to about 10mg/kg. An indicated daily dosage in the larger mammal, including, butnot limited to, humans, is in the range from about 0.5 mg to about 1000mg, conveniently administered in a single dose or in divided doses,including, but not limited to, up to four times a day or in extendedrelease form. Suitable unit dosage forms for oral administration includefrom about 1 to about 500 mg active ingredient. In one embodiment, theunit dosage is about 1 mg, about 5 mg, about, 10 mg, about 20 mg, about50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, or about500 mg. The foregoing ranges are merely suggestive, as the number ofvariables in regard to an individual treatment regime is large, andconsiderable excursions from these recommended values are not uncommon.Such dosages may be altered depending on a number of variables, notlimited to the activity of the compound used, the disease or conditionto be treated, the mode of administration, the requirements of theindividual subject, the severity of the disease or condition beingtreated, and the judgment of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD₅₀ and ED₅₀. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Combination Treatments

The compounds of Formula (I), (II), (IIA) or (IIB), and compositionsthereof, may also be used in combination with other well knowntherapeutic agents that are selected for their therapeutic value for thecondition to be treated. In general, the compositions described hereinand, in embodiments where combinational therapy is employed, otheragents do not have to be administered in the same pharmaceuticalcomposition, and may, because of different physical and chemicalcharacteristics, have to be administered by different routes. Thedetermination of the mode of administration and the advisability ofadministration, where possible, in the same pharmaceutical composition,is well within the knowledge of the skilled clinician. The initialadministration can be made according to established protocols known inthe art, and then, based upon the observed effects, the dosage, modes ofadministration and times of administration can be modified by theskilled clinician.

In certain instances, it may be appropriate to administer at least onecompound of Formula (I), (II), (IIA) or (IIB) described herein incombination with another therapeutic agent. By way of example only, ifone of the side effects experienced by a patient upon receiving one ofthe compounds herein, such as a compound of Formula (I), (II), (IIA) or(IIB), is nausea, then it may be appropriate to administer ananti-nausea agent in combination with the initial therapeutic agent. Or,by way of example only, the therapeutic effectiveness of one of thecompounds described herein may be enhanced by administration of anadjuvant (i.e., by itself the adjuvant may have minimal therapeuticbenefit, but in combination with another therapeutic agent, the overalltherapeutic benefit to the patient is enhanced). Or, by way of exampleonly, the benefit experienced by a patient may be increased byadministering one of the compounds described herein with anothertherapeutic agent (which also includes a therapeutic regimen) that alsohas therapeutic benefit. In any case, regardless of the disease,disorder or condition being treated, the overall benefit experienced bythe patient may simply be additive of the two therapeutic agents or thepatient may experience a synergistic benefit.

The particular choice of compounds used will depend upon the diagnosisof the attending physicians and their judgment of the condition of thepatient and the appropriate treatment protocol. The compounds may beadministered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the disease, disorder, or condition, thecondition of the patient, and the actual choice of compounds used. Thedetermination of the order of administration, and the number ofrepetitions of administration of each therapeutic agent during atreatment protocol, is well within the knowledge of the skilledphysician after evaluation of the disease being treated and thecondition of the patient.

Therapeutically-effective dosages can vary when the drugs are used intreatment combinations. Methods for experimentally determiningtherapeutically-effective dosages of drugs and other agents for use incombination treatment regimens are described in the literature. Forexample, the use of metronomic dosing, i.e., providing more frequent,lower doses in order to minimize toxic side effects, has been describedextensively in the literature Combination treatment further includesperiodic treatments that start and stop at various times to assist withthe clinical management of the patient.

For combination therapies described herein, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the disease orcondition being treated and so forth. In addition, when co-administeredwith one or more biologically active agents, the compound providedherein may be administered either simultaneously with the biologicallyactive agent(s), or sequentially. If administered sequentially, theattending physician will decide on the appropriate sequence ofadministering protein in combination with the biologically activeagent(s).

In any case, the multiple therapeutic agents (one of which is a compoundof Formula (I), (II), (IIA) or (IIB) described herein) may beadministered in any order or even simultaneously. If simultaneously, themultiple therapeutic agents may be provided in a single, unified form,or in multiple forms (by way of example only, either as a single pill oras two separate pills). One of the therapeutic agents may be given inmultiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than four weeks. In addition, the combinationmethods, compositions and formulations are not to be limited to the useof only two agents; the use of multiple therapeutic combinations arealso envisioned.

It is understood that the dosage regimen to treat, prevent, orameliorate the condition(s) for which relief is sought, can be modifiedin accordance with a variety of factors. These factors include thedisorder or condition from which the subject suffers, as well as theage, weight, sex, diet, and medical condition of the subject. Thus, thedosage regimen actually employed can vary widely and therefore candeviate from the dosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapydisclosed herein may be a combined dosage form or in separate dosageforms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy may also beadministered sequentially, with either therapeutic compound beingadministered by a regimen calling for two-step administration. Thetwo-step administration regimen may call for sequential administrationof the active agents or spaced-apart administration of the separateactive agents. The time period between the multiple administration stepsmay range from, a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent. Circadian variation of the target moleculeconcentration may also determine the optimal dose interval.

In addition, the compounds described herein also may be used incombination with procedures that may provide additional or synergisticbenefit to the patient. By way of example only, patients are expected tofind therapeutic and/or prophylactic benefit in the methods describedherein, wherein pharmaceutical composition of a compound disclosedherein and/or combinations with other therapeutics are combined withgenetic testing to determine whether that individual is a carrier of amutant gene that is known to be correlated with certain diseases orconditions.

The compounds described herein and combination therapies can beadministered before, during or after the occurrence of a disease orcondition, and the timing of administering the composition containing acompound can vary. Thus, for example, the compounds can be used as aprophylactic and can be administered continuously to subjects with apropensity to develop conditions or diseases in order to prevent theoccurrence of the disease or condition. The compounds and compositionscan be administered to a subject during or as soon as possible after theonset of the symptoms. The administration of the compounds can beinitiated within the first 48 hours of the onset of the symptoms,preferably within the first 48 hours of the onset of the symptoms, morepreferably within the first 6 hours of the onset of the symptoms, andmost preferably within 3 hours of the onset of the symptoms. The initialadministration can be via any route practical, such as, for example, anintravenous injection, a bolus injection, infusion over about 5 minutesto about 5 hours, a pill, a capsule, transdermal patch, buccal delivery,and the like, or combination thereof A compound is preferablyadministered as soon as is practicable after the onset of a disease orcondition is detected or suspected, and for a length of time necessaryfor the treatment of the disease, such as, for example, from 1 day toabout 3 months. The length of treatment can vary for each subject, andthe length can be determined using the known criteria. For example, thecompound or a formulation containing the compound can be administeredfor at least 2 weeks, preferably about 1 month to about 5 years.

Inhibitors of SOCE

In one aspect, compounds of Formula (I), (II), (IIA) or (IIB) may beadministered or used in conjunction with other known inhibitors of SOCE.In one aspect, the known inhibitors of SOCE are non-selectiveinhibitors. In one aspect, the known inhibitors of SOCE are selectiveinhibitors.

A variety of inhibitors of SOCE have been described. Known inhibitors ofSOCE include:

-   a) Cations, which include lanthanide cations, such as for example,    Gd³⁺, La³⁰ ;-   b) P-450 inhibitors, which include econazole, miconazole,    clotrimazole, ketoconazole;-   c) Cyclooxygenase inhibitors, which include niflumic acid,    flufenamic acid, tenidap;-   d) Lipoxygenase inhibitors, which include nordihydroguaiaretic acid,    eicosatetraynoic acid;-   e) Compounds that are channel blockers, which include SK&F 96365,    SC38249, LU52396, L-651,582, tetrandrine, 2-APB;-   f) Compounds that inhibit SOCE not by an action on the SOC channels    themselves, which include U73122 (phospholipase C inhibitor),    wortmannin (phosphatidylinositol kinase inhibitor).

Some of these known inhibitors of SOCE have non-specific actions and/ormultiple modes of action that contribute to the inhibition of SOCE,which include blocking the pore of the SOC channel (Channel blockers),inhibition of mitochondrial ATP synthesis that appears to support SOCE(Gamberucci et al., J Biol. Chem., 269, 23597-23602, 1994; Marriott etal., Am. J. Physiol., 269, C766-C774, 1995), disturbances of cytoplasmicpH (Muallem et al., Am. J. Physiol., 257, G917-G924, 1989), as well asinhibiting the activation of SOC channels.

Immunosuppresants

In one embodiment, compounds of Formula (I), (II), (IIA) or (IIB) areadministered as single agents in immunosuppressive therapy to reduce,inhibit, or prevent activity of the immune system. Immunosuppressivetherapy is clinically used to: prevent the rejection of transplantedorgans and tissues (e.g. bone marrow, heart, kidney, liver); treatmentof autoimmune diseases or diseases that are most likely of autoimmuneorigin (e.g. rheumatoid arthritis, myasthenia gravis, systemic lupuserythematosus, Crohn's disease, and ulcerative colitis); and treatmentof some other non-autoimmune inflammatory diseases (e.g. long termallergic asthma control).

In some embodiments, a compound of Formula (I), (II), (IIA) or (IIB) maybe administered with other immunosuppresants selected from among:Calcineurin inhibitors (such as, but not limited to, cyclosporin,tacrolimus); mTOR inhibitors (such as, but not limited to, sirolimus,everolimus); anti-proliferatives (such as, but not limited to,azathioprine, mycophenolic acid); corticosteroids (such as, but notlimited to, prednisone, cortisone acetate, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone acetate, deoxycorticosterone acetate,aldosterone, hydrocortisone); antibodies (such as, but not limited to,monoclonal anti-IL-2Rα receptor antibodies (basiliximab, daclizumab),polyclonal anti-T-cell antibodies (anti-thymocyte globulin (ATG),anti-lymphocyte globulin (ALG))).

Other immunosuppresants include, but are not limited to: glucocorticoids(alclometasone, aldosterone, amcinonide, beclometasone, betamethasone,budesonide, ciclesonide, clobetasol, clobetasone, clocortolone,cloprednol, cortisone, cortivazol, deflazacort, deoxycorticosterone,desonide, desoximetasone, desoxycortone, dexamethasone, diflorasone,diflucortolone, difluprednate, fluclorolone, Fludrocortisone,fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone,fluprednidene, fluticasone, formocortal, halcinonide, halometasone,hydrocortisone/cortiso1, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, medrysone,meprednisone, methylprednisolone, methylprednisolone aceponate,mometasone furoate, paramethasone, prednicarbate, prednisone,prednisolone, prednylidene, rimexolone, tixocortol, triamcinolone,ulobetasol), cyclophosphamide, nitrosoureas, cisplatin, carboplatin,oxaliplatin, methotrexate, azathioprine, mercaptopurine, pyrimidineanalogues, protein synthesis inhibitors, methotrexate, azathioprine,mercaptopurine, dactinomycin, anthracyclines, mitomycin C, bleomycin,mithramycin, Atgam®, Thymoglobuline®, OKT3®, basiliximab, daclizumab,cyclosporin, tacrolimus, sirolimus, Interferons (IFN-β, IFN-γ), opioids,TNF binding proteins (infliximab, etanercept, adalimumab, golimumab),mycophenolic acid, mycophenolate mofetil, FTY720, as well as thoselisted in U.S. Pat. No. 7,060,697.

Agents for Treating Autoimmune Diseases, Inflammatory Diseases

Where the subject is suffering from or at risk of suffering from anautoimmune disease, disorder or condition, or an inflammatory disease,disorder or condition, a compound of Formula (I), (II), (IIA) or (IIB)may be administered in any combination with one or more of the followingtherapeutic agents: immunosuppressants (e.g., tacrolimus, cyclosporin,rapamicin, methotrexate , cyclophosphamide, azathioprine,mercaptopurine, mycophenolate, or FTY720), glucocorticoids (e.g.,prednisone, cortisone acetate, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisone acetate, deoxycorticosterone acetate, aldosterone),non-steroidal anti-inflammatory drugs (e.g., salicylates, arylalkanoicacids, 2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs,or sulphonanilides), Cox-2-specific inhibitors (e.g., valdecoxib,etoricoxib, lumiracoxib, celecoxib, or rofecoxib), leflunomide, goldthioglucose, gold thiomalate, aurofin, sulfasalazine,hydroxychloroquinine, minocycline, TNF-α binding proteins (e.g.,infliximab, etanercept, or adalimumab), abatacept, anakinra,interferonβ, interferon-γ, interleukin-2, antileukotrienes,theophylline, or anticholinergics.

In one embodiment, compounds of Formula (I), (II), (IIA) or (IIB)described herein, may be administered in combination with inhibitors ofNFAT-calcineurin pathway. In one embodiment, the inhibitors ofNFAT-calcineurin pathway include, but are not limited to, Cyclosporin A(CsA) and tacrolimus (FK506).

In one embodiment, a compound of Formula (I), (II), (IIA) or (IIB), orcompositions and medicaments that include a compound of Formula (I),(II), (IIA) or (IIB), may be administered to a patient in combinationwith an anti-inflammatory agent including, but not limited to,non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids(glucocorticoids).

NSAIDs include, but are not limited to: aspirin, salicylic acid,gentisic acid, choline magnesium salicylate, choline salicylate, cholinemagnesium salicylate, choline salicylate, magnesium salicylate, sodiumsalicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium,fluorobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac, ketorolactromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin,sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid,piroxicam, meloxicam, COX-2 specific inhibitors (such as, but notlimited to, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib,lumiracoxib, CS-502, JTE-522, L-745,337 and NS398).

Combinations with NSAIDs, which are selective COX-2 inhibitors, arecontemplated herein. Such compounds include, but are not limited tothose disclosed in U.S. Pat. No. 5,474,995; U.S. Pat. No. 5,861,419;U.S. Pat. No. 6,001,843; U.S. Pat. No. 6,020,343, U.S. Pat. No.5,409,944; U.S. Pat. No. 5,436,265; U.S. Pat. No. 5,536,752; U.S. Pat.No. 5,550,142; U.S. Pat. No. 5,604,260; U.S. Pat. No. 5,698,584; U.S.Pat. No. 5,710,140; WO 94/15932; U.S. Pat. No. 5,344,991; U.S. Pat. No.5,134,142; U.S. Pat. No. 5,380,738; U.S. Pat. No. 5,393,790; U.S. Pat.No. 5,466,823; U.S. Pat. No. 5,633,272; U.S. Pat. No. 5,932,598 and6,313,138; all of which are hereby incorporated by reference.

Compounds that have been described as selective COX-2 inhibitors and aretherefore useful in the methods or pharmaceutical compositions describedherein include, but are not limited to, celecoxib, rofecoxib,lumiracoxib, etoricoxib, valdecoxib, and parecoxib, or apharmaceutically acceptable salt thereof.

Corticosteroids, include, but are not limited to: betamethasone,prednisone, alclometasone, aldosterone, amcinonide, beclometasone,betamethasone, budesonide, ciclesonide, clobetasol, clobetasone,clocortolone, cloprednol, cortisone, cortivazol, deflazacort,deoxycorticosterone, desonide, desoximetasone, desoxycortone,dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone,fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone,fluperolone, fluprednidene, fluticasone, formocortal, halcinonide,halometasone, hydrocortisone/cortiso1, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,medrysone, meprednisone, methylprednisolone, methylprednisoloneaceponate, mometasone furoate, paramethasone, prednicarbate,prednisone/prednisolone, rimexolone, tixocortol, triamcinolone, andulobetasol.

Other agents used as anti-inflammatories include those disclosed in U.S.patent publication 2005/0227929, herein incorporated by reference.

Some commercially available anti-inflammatories include, but are notlimited to: Arthrotec® (diclofenac and misoprostol),Asacol®(5-aminosalicyclic acid), Salofalk® (5-aminosalicyclic acid),Auralgan® (antipyrine and benzocaine), Azulfidine® (sulfasalazine),Daypro® (oxaprozin), Lodine(etodolac), Ponstan® (mefenamic acid),Solumedrol® (methylprednisolone), Bayer®(aspirin), Bufferin® (aspirin),Indocin® (indomethacin), Vioxx® (rofecoxib), Celebrex® (celecoxib),Bextra® (valdecoxib), Arcoxia® (etoricoxib), Prexige® (lumiracoxib),Advil®, Motrin® (ibuprofen), Voltaren®(diclofenac), Orudis®(ketoprofen),Mobic®(meloxicam), Relafen® (nabumetone), Aleve®, Naprosyn® (naproxen),Feldene® (piroxicam).

In one embodiment, compounds of Formula (I), (II), (IIA) or (IIB) areadministered in combination with leukotriene receptor antagonistsincluding, but are not limited to, BAY u9773 (see EP 00791576; published27 Aug. 1997), DUO-LT (Tsuji et al, Org. Biomol. Chem., 1, 3139-3141,2003), zafirlukast (Accolate®), montelukast (Singulair®), prankulast(Onon®), and derivatives or analogs thereof.

Kits/Articles of Manufacture

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. Such kits can includea carrier, package, or container that is compartmentalized to receiveone or more containers such as vials, tubes, and the like, each of thecontainer(s) including one of the separate elements to be used in amethod described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical productsinclude, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,containers, syringes, bottles, and any packaging material suitable for aselected formulation and intended mode of administration and treatment.A wide array of formulations of the compounds and compositions providedherein are contemplated as are a variety of treatments for any disease,disorder, or condition that would benefit by inhibition of CRAC channelactivity.

For example, the container(s) can include one or more compoundsdescribed herein, optionally in a composition or in combination withanother agent as disclosed herein. The container(s) optionally have asterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). Such kits optionally comprising a compound with anidentifying description or label or instructions relating to its use inthe methods described herein.

A kit will typically may include one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of a compound described herein. Non-limiting examplesof such materials include, but not limited to, buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use. A set of instructions will alsotypically be included.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions can be presentedin a pack or dispenser device which can contain one or more unit dosageforms containing a compound provided herein. The pack can for examplecontain metal or plastic foil, such as a blister pack. The pack ordispenser device can be accompanied by instructions for administration.The pack or dispenser can also be accompanied with a notice associatedwith the container in form prescribed by a governmental agencyregulating the manufacture, use, or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of the drugfor human or veterinary administration. Such notice, for example, can bethe labeling approved by the U.S. Food and Drug Administration forprescription drugs, or the approved product insert. Compositionscontaining a compound provided herein formulated in a compatiblepharmaceutical carrier can also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition.

Assays

Several techniques may be used to evaluate store operated calcium entryand calcium signalling in cells. Such techniques include, but are notlimited to, patch clamp electrophysiology (measurement of calcium ionsor other ions across cell membranes, such as plasma membranes),capacitance measurements (allows exocytosis to be followed at the levelof single cells), calcium imaging using fluorescent dyes allows patternsof calcium movement within the cytoplasm to be tracked, fluorescenceresonance energy transfer (FRET) enables protein-protein interactions tobe evaluated, and molecular biology methods allow for the manipulationof the levels of expression of proteins of interest.

A wide variety of assay methods may be used to examine the modulation ofintracellular calcium by compounds of Formula (I), (II), (IIA) and(IIB). Such assays include in vitro cell based assays as well as in vivoanimal models. Any assays that detect, monitor or measure an effect onintracellular calcium, including calcium entry-mediated events can beused. Such assays include, but are not limited to, assays monitoring,measuring and/or detecting intracellular calcium levels, modulation ofcalcium levels, and movement of calcium into, out of or within cells andintracellular organelles. Assays can also include monitoring, measuringand/or detecting calcium entry-mediated events and molecules involved incalcium entry-mediated events such as, but not limited to, signaltransduction molecules, transcription factors, secreted molecules andother molecules that are affected by changes in calcium homeostasis.Assays include, but are not limited to, those described herein and thosedescribed in US patent publication no. 2007/0031814 and WO 07/081804,herein incorporated by reference.

Cells and Cell Models

For in vitro testing of the modulation of intracellular calcium bycompounds of Formula (I), (II), (IIA) and (IIB), a wide variety of celltypes for such assays are available. In a particular embodiment, thecell is one in which store-operated calcium entry occurs or that can bemanipulated such that store-operated calcium entry occurs in the cell.In particular embodiments, the cell contains one or more proteinsinvolved in modulating intracellular calcium (and, in particular, isinvolved in, participates in and/or provides for store-operated calciumentry, movement of calcium into, out of or within an intracellularorganelle or calcium store, modulation of calcium levels in anintracellular organelle or calcium store (e.g., endoplasmic reticulum)and/or calcium buffering), such as those provided herein. In particularembodiments, the protein(s) include a STIM proteins (including STIM1,STIM2, DSTIM and CSTIM protein) and/or Orai proteins (Orai1, Orai2,Orai3). The cell may endogenously express the protein(s) orrecombinantly express the protein(s).

Cells for use in the methods may be of any species. In one embodiment,the cells can be eukaryotic cells. In one embodiment, the cells can beyeast, insect (e.g., Drosophila or Anopheles), or mammalian cells.Mammalian cells include, but are not limited to, rodent (e.g., mouse,rat and hamster), primate, monkey, dog, bovine, rabbit and human cells.A variety of cell types can be used in the methods, including, forexample, neuronal, nervous system, brain, immune system cells, e.g., Tlymphocytes and B cells, primary cells, blood and hematopoietic cells,stromal cells, myeloid cells, lymphoid cells, and a variety of tumor andcancer cells. Particular cells include Drosophila Schneider 2 or S2cells, human embryonic kidney (HEK293) cells, rat basophilic leukemia(RBL-2H3) cells, Jurkat cells, epithelial cells, rhabdomyosarcoma cells,rhabdoid cells, retinoblastoma cells, neuroepithelioma cells,neuroblastoma cells, osteosarcoma cells, fibroblasts, bone marrow stromacells, erythroleukemia cells and lymphoblast cells. Other cell linesinclude HEK 293 and 293T, CHO (including CHO-Ki), LTK-, N2A, H6, andHGB. Many such cells and cell lines are available through celldepositories such as, for example, the American Type Culture Collection(ATCC, Manassas, Va.). Primary cells can be obtained by isolation fromtissue sources. The generation, maintenance and use of such cells andcell lines is well known.

Cells from a known cell line can be used, such as neuroblastoma SH-SY5Ycells, pheochromocytoma PC12 cells, neuroblastoma SK-N-BE(2)C or SK-N-SHcells, human SK-N-MC neuroepithelioma cells, SMS-KCNR cells, human LAN-5neuroblastoma cells, human GI-CA-N neuroblastoma cells, human GOTOneuroblastoma cells, mouse Neuro 2a (N2A) neuroblastoma cells and/orhuman IMR 32 neuroblastoma cells, chronic myeloid leukemia cells (e.g.,human K562 cells), promyelocytic leukemia cells (e.g., HL60 cells) andhistiocytic lymphoma cells (e.g., U937 cells), Burkitt's lymphoma cells(e.g., CA46 cells), B-cells (e.g., NALM6), acute lymphoblastic leukemiacells (e.g., MOLT4 cells), T cells (e.g. Jurkat cells) and early T-ALL(e.g., DU528) cells.

The choice of a cell for use in an in vitro assay to test the modulationof intracellular calcium by compounds of Formula (I), (II), (IIA) or(IIB) can involve several considerations, including, for example, aparticular protein that is being used in the method and a particularaspect or activity of intracellular calcium modulation that is beingmonitored or assessed in the method.

In one embodiment, the modulation of intracellular calcium by a compoundof Formula (I), (II), (IIA) or (IIB) is examined by monitoring orassessing the effect on store-operated calcium entry. Cells typicallyused in such methods exhibit store-operated calcium entry eithernaturally or through manipulation of the cells. Cells that endogenouslyexhibit store-operated calcium entry include some excitable cells andmost non-excitable cells and can be identified using methods describedherein and/or known in the art.

In one embodiment, it may be desirable to utilize a cell that containscomponents of signaling and messenger systems that can effect release ofcalcium from intracellular stores. For example, cells containingcomponents of receptor-mediated phospholipase C (PLC) activation systemscan be used for physiological activation (via generation of IP₃) ofstore depletion to facilitate monitoring of store-operated calciumentry. Receptor-mediated PLC activation occurs through distinct couplingmechanisms: PLC-β activation by G protein-coupled receptors (GPCRs) andPLC-γ activation by tyrosine kinase receptors and nonreceptor tyrosinekinases. Thus, cells containing a receptor-mediated PLC-activationsystem can be monitored or assessed for store-operated calcium entryupon agonist activation of one or more receptors known to participate inthe system. (see e.g. Bouron (2000) FEBS Lett 470:269-272; Millar et al.(1995) J. Exp. Biol. 198:1843-1850; Yagodin et al. (1998) Cell Calcium23:219-228; Yagodin et al. (1999) Cell Calcium 25:429-438; and Pattersonet al. (2002) Cell 111:1-20).

An assessment of intracellular calcium after treatment with a compoundof Formula (I), (II), (IIA) or (IIB) can be made under a variety ofconditions. Conditions can be selected to evaluate the effect of testagent on a specific aspect of intracellular calcium. For example,reagents and conditions are known, and can be used, for specificallyevaluating store-operated calcium entry, resting cytosolic calciumlevels, calcium buffering, and calcium levels of and calcium uptake byor release from intracellular organelles. Resting cytosolic calciumlevels, intracellular organelle calcium levels and cation movement maybe assessed using any of the methods described herein or known in theart. Such methods of assessing modulation in intracellular calciuminclude, but are not limited to, calcium-sensitive indicator-basedmeasurements, such as fluo-3, mag-fura 2 and ER-targeted aequorin,labelled calcium (such as ⁴⁵Ca²⁺)-based measurements, andelectrophysiological measurements. Particular aspects of ion flux thatmay be assessed include, but are not limited to, a reduction (includingelimination) in the amount of ion flux, altered biophysical propertiesof the ion current, and altered sensitivities of the flux to activatorsor inhibitors of calcium flux processes, such as, for example,store-operated calcium entry. Reagents and conditions for use inspecifically evaluating receptor-mediated calcium movement and secondmessenger-operated calcium movement are also available.

Evaluation of Store-Operated Calcium Entry

In one aspect, compounds of Formula (I), (II), (IIA) or (IIB) are addedto cells under conditions that permit store-operated calcium entry tooccur in order to assess the effects of Formula (I), (II), (IIA) or(IIB) on store-operated calcium entry. Such conditions are describedherein and are known in the art.

For example, in one method cells may be treated to reduce the calciumlevels of intracellular calcium stores and then analyzed for evidence ofion (e.g., calcium) influx in response thereto in the presence of acompound of Formula (I), (II), (IIA) or (IIB). Techniques for reducingcalcium levels of intracellular stores and for analyzing cells forevidence of ion (e.g., calcium) influx are known in the art anddescribed herein.

In other methods, electrophysiological analysis of currents across acell-detached plasma membrane patch or an outside-out membrane vesiclemay be used to detect or monitor store-operated channel currents (e.g.,I_(SOC), I_(CRAC)) in the presence of a compound of Formula (I), (II),(IIA) or (IIB).

Evaluation of Calcium Entrv-Mediated Events

A number of molecules involved in calcium-regulated pathways are known.Evaluation of molecules involved in calcium-entry mediated events can beused to monitor intracellular calcium, and can be used, for example inscreening assays described herein to monitor the effects of compounds ofFormula (I), (II), (IIA) and (IIB). Examples of assays include but arenot limited to assays which detect, or determine the presence, levels,alteration of levels, production, modification (such as phosphorylationand dephosphorylation), translocation, degradation and activity ofmolecules involved in calcium-entry mediated events (see for example,Trevillyan et al. (2001) J. Biol. Chem. 276:48118-26). The assaysdescribed herein can be used with cells that have been treated with orcontacted with a compound of Formula (I), (II), (IIA) or (IIB), or thatexpress an altered amount of a test molecule (such as a protein involvedin calcium regulation, including a STIM protein, Orai protein), or withcontrol cells. The assays can also be conducted in cells that have beenstimulated with a physiological or non-physiological activator, or inunstimulated cells. The following are representative assays formolecules involved in calcium-entry mediated events and are meant to beexemplary only. Other assays for these molecules and assays for othermolecules involved in calcium-entry mediated events can also be employedin any of the screening and/or modulation methods described herein.

β-hexosaminidase Release

In mast cells, Ca²⁺ influx results in degranulation and release ofinflammatory mediators such as heparin, histamine and enzymes such asβ-hexosaminidase. Detecting and/or measuring release of such moleculescan thus be used to monitor intracellular calcium. For example, mediafrom mast cells can be collected. A suitable substrate forβ-hexosaminidase (e.g. p-nitrophenyl-acetyl-glucosamide) can then beadded and the absorbance of the resulting mixture assessed to measurethe relative amount of β-hexosaminidase activity in the samples (Funabaet al. (2003) Cell Biol. International 27:879-85).

Calcium/Calmodulin-Dependent CaN Phosphatase Activity

The phosphatase calcineurin (CaN) dephosphorylates various proteins,affecting their activity and localization. CaN activity can be assessedby incubating purified CaN and a CaN substrate, for example aradiolabeled peptide corresponding to a sequence in the RH subunit ofcAMP-dependent kinase, either with or without a compound of Formula (I),(II), (IIA) or (IIB) (see, Trevillyan et al. (2001) J. Biol. Chem276:48118-26). The level of radiolabeled peptide and/or the amount offree inorganic phosphate released can be measured to assess CaNdephosphorylation activity.

NFAT Transcriptional Activity

The NFAT (nuclear factor of activated T cells) transcription factorregulates a number of genes in response to intracellular calcium levels.For example, NFAT proteins regulate the transcription of cytokine genesinvolved in the immune response. Promoters from NFAT-regulated genes,and/or regulatory regions and elements from these genes, can be used tomonitor NFAT regulated expression and thereby monitor intracellularcalcium. Reporter gene fusions can be constructed with NFAT regulatedpromoters or NFAT-regulated elements operably linked to a reporter genesuch as luciferase, β-galactosidase, green fluorescent protein (GFP) orany other known reporter in the art (see for example, Published U.S.Application no. 2002-0034728). The amount of reporter protein oractivity is a measure of NFAT activity.

NFAT Phosphorylation

NFAT activation is regulated primarily through its phosphorylation,which in turn regulates its subcellular localization. In unstimulatedcells, NFAT is a hyperphosphorylated cytosolic protein. An elevation inintracellular Ca²⁺, induced by a variety of mechanisms, increases theactivity of the Ca²⁺-calmodulin-dependent phosphatase, calcineurin.Activated calcineurin dephosphorylates multiple serine residues withinthe regulatory region of the NFAT molecule. NFAT is rephosphorylated inresponse to decreases in Ca²⁺ levels or CaN inhibition.

The phosphorylation state of NFAT can be monitored for example, byexpressing a detectably tagged NFAT protein in cells, such as a His6tagged-NFAT. Tagged NFAT can be purified from cells using Ni²⁺chromatography and subjected to gel electrophoresis and staining orwestern blotting. More highly phosphorylated forms of NFAT can bedistinguished by their slower migration. The state of phosphorylatedNFAT can be used as a measure of NFAT activation (see, Trevillyan et al.(2001) J. Biol. Chem 276:48118-26).

NFAT Nuclear Localization

NFAT localization between the cytoplasm and nucleus is regulated by thephosphorylation state of NFAT. Phosphorylation of NFAT prevents nuclearlocalization by masking the nuclear localization sequence. NFAT nuclearlocalization can be monitored, for example, by expressing fluorescentlytagged NFAT, for example, GFP-NFAT, in cells. Confocal microscopy can beused to monitor nuclear localization of the tagged NFAT (see, Trevillyanet al. (2001) J. Biol. Chem 276:48118-26).

Cytokine Secretion

Cytokine secretion, such as IL-2 secretion, can be monitored usingprotein detection assays. For example, supernatant can be collected fromimmune cells. An ELISA assay or other suitable format with IL-2antibodies can be used to detect and/or measure the amount of IL-2secreted as compared to control cells. Secretion of other cytokines, forexample, TNF-α, can also be detected in similar assays.

Cytokine Expression

Expression of cytokines, such as, but not limited to IL-2, can beassessed either directly or indirectly in cells. For example, inindirect methods, an IL-2 promoter can be operably linked to a reportergene such as luciferase or β-galactosidase, and the reporter constructintroduced into cells. Reporter gene expression can be monitored andcompared to gene expression in control cells (see, Trevillyan et al.(2001) J. Biol. Chem 276:48118-26). Alternatively, expression ofendogenous or recombinant IL-2 mRNA or protein can be assessed.

T Cell Proliferation

Cytokines such as IL-2 are necessary for T-cell proliferation inresponse to mitogen or alloantigen stimulation, and thus T-cellproliferation is altered by changes in cytokine expression or secretion.T cells can be induced, such as with concanavalin A or alloreactivelymphocytes and T cell proliferation measured, for example, bysubjecting cells to a pulse of ³H-thymidine and measuring ³H-thymidineincorporation (see, Trevillyan et al. (2001) J. Biol. Chem276:48118-26).

The modulation (e g inhibition or reduction) of SOCE by compounds ofFormula (I), (II), (IIA) or (IIB) may be determined by evaluation of anyof the following criteria:

-   a. there is direct inhibition of increased [Ca²⁺]i as measured by a    calcium indicator;-   b. there is a direct inhibition of I_(SOC) or I_(CRAC) as measured    by patch clamp;-   c. there is inhibition of downstream signaling functions such as    calcineurin activity, NFAT subcellular localization, NFAT    phosphorylation, and/or cytokine, e.g., IL-2, production; or-   d. there are modifications in activation-induced cell proliferation,    differentiation and/or apoptotic signaling pathways.

Animal Models

Animal models that can be used in embodiments of the methods furtherinclude animals, such as, but not limited to non-human animals, whichhave, in at least some of their cells, an alteration or defect in, oraberrant functioning of, a cellular process which relies on or isregulated by intracellular calcium. Cellular processes that rely on orare regulated by intracellular calcium include, for example, cellularactivation, gene expression, cellular trafficking, and apoptosis.Diseases/disorders that involve defects that may be at least partiallycompensated for by modulation of intracellular calcium include, but arenot limited to: autoimmune disorders, including rheumatoid arthritis,inflammatory bowel disease, Sjogren's syndrome (cytokines associatedwith lymphocyte invasion of salivary epithelial cells can reduce calciummobilization in parotid cells; also, T-cell activation, includingactivation of transcription factors, cytokine gene expression and cellproliferation, depends on sustained elevation of intracellular calciumlevel provided by store-operated calcium influx), asthma (store-operatedcalcium entry may play an important role in mediating bronchialchonstriction and bronchial smooth muscle cell proliferation),glomerulonephritis and glomerular inflammation (changes in intracellularcalcium, such as by store-operated calcium entry, signal monocyteadhesion in a co-culture model of glomerular inflammation).

Types of animal models include, but are not limited to, non-humananimals, such as non-human invertebrates and vertebrates and non-humanmammals, rodents (e.g., mice, rat and hamster), cows, chickens, pigs,goats, dogs, sheep, insects, Drosophila, nematodes, worms, C. elegans,monkeys, gorillas, and other primates.

Animal models include transgenic and non-transgenic animals. One exampleof such an animal model that can be used in particular embodiments ofthe methods is a rodent model of airway hyperresponsiveness (AHR), acharacteristic of asthma. This model can be generated, for example, bysensitization through immunization with ovalbumin followed by exposureto aerosolized ovalbumin and challenge by cholinergic stimulation (e.g.,via administration of methacholine or acetylcholine) (see, e.g., Xu etal. (2002) J. Appl. Physiol. 93:1833-1840; Humbles et at (2002) Proc.Natl. Acad. Sci. 99:1479-1484). Airway hyperresponsiveness (which can beevaluated using methods known in the art, e.g., using barometricplethysmography to record respiratory pressure curves and throughmeasurement of pulmonary parameters such as pulmonary conductance andpulmonary compliance) can be assessed and compared in animals treatedand not treated with a compound of Formula (I), (II), (IIA) or (IIB). Afurther example of an animal model that can be used in particularembodiments of the methods is a rodent model of mesangial proliferativeglomerulonephritis, which can be generated, for example, byadministration of anti-Thyl.1 antibody (see, e.g., Jefferson and Johnson(1999) J. Nephrol. 12:297-307). Any number of parameters indicative ofglomerulonephritis or renal dysfunction (e.g., mesangial cellproliferation, blood pressure, urinary protein excretion, creatinineclearance, glomerulosclerosis index and other parameters) can beevaluated and compared in animals treated with and not treated with testagent. The non-obese diabetic (NOD) mouse, an inbred mouse strain thatspontaneously develops an autoimmune diabetes that shares manyimmunogenetic features with Type 1 diabetes mellitus, is another exampleof an animal model that can be used in a particular embodiment of themethods. These mice also manifest many characteristics of autoimmuneexocrinopathy (such as Sjorgen's syndrome) including declining exocrinetissue secretory function (see, e.g., Humphreys-Beher and Peck (1999)Arch. Oral Biol. 44 Suppl 1:S21-25 and Brayer et al. (2000) J Rheumatol.27:1896-1904). Characteristics relevant to Sjorgen's syndrome (e.g.,lymphocytic infiltrates in exocrine glands (e.g., salivary and lacrimalglands), presence of dendritic cells and macrophages in submandibularglands, integrity of the lacrimal gland by measurement of basal andstimulated tear secretion, saliva flow rates and amylase activity) canbe evaluated and compared in animals treated with and not treated with acompound of Formula (I), (II), (IIA) or (IIB). An animal (e.g., rodent)model of autoimmune disease can also be used in particular embodimentsof the methods. Such animals include rat models available through theNational Institutes of Health (NIH) Autoimmune Rat Model Repository andDevelopment Center (Bethesda, Md.; accessible atwww.ors.od.nih.gov/dirs/vrp/ratcenter). One rat model of rheumatoidarthritis (RA) and related chronic/inflammatory autoimmune diseases isthe collagen-induced arthritis (CIA) model (see, e.g., Griffiths andRemmers (2001) Immunol. Rev. 184:172-183). Characteristic phenotypes ofautoimmune disease (e.g. altered levels of immune reactivity toself-antigens, chronic inflammation of autoantigen-expressing targetorgans, and activation and participation of invading mononuclear cellsand tissue fibroblasts in organ damage) can be evaluated and compared inanimals treated with and not treated with a compound of Formula (I),(II), (IIA) or (IIB). An animal (e.g., rodent) model of neuropathic orinflammatory pain can also be used in a particular embodiment of themethods. For example, one rat model of neuropathic pain involvesdevelopment of tactile allodynia (exaggerated response to otherwiseinnocuous stimuli) after ligation of lumbar spinal nerves (see, e.g.,Chaplan et al. (1994) J. Neurosci. Methods 53:55-63 and Luo et al.(2001) J. Neurosci. 21:1868-1875). Tactile allodynia, one characteristicfeature of neuropathic pain, can be evaluated (e.g., by evaluating pawwithdrawal threshold in response to application of pressure) andcompared in animals treated and not treated with a compound of Formula(I), (II), (IIA) or (IIB).

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein. The starting materialsand reagents used for the synthesis of the compounds described hereinmay be synthesized or can be obtained from commercial sources, such as,but not limited to, Sigma-Aldrich, Acros Organics, Fluka, and FischerScientific.

Example 1 2-(4-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylicacid (Compound 1)

Elemental sulfur (10 mmol), ethyl cyanoacetate (10 mmol), andp-bromo-acetophenone (10 mmol), ethanol (2 mL), and morpholine (2 mL)are mixed together and stirred at room temperature for approximately 20hours. The reaction mixture is poured into 20 mL of methylene chlorideand 25 mL of brine. The layers are separated, and the aqueous layer isextracted with 2×15 mL methylene chloride. The organic layers arecombined, dried over anhydrous sodium sulfate, filtered and concentratedto dryness. The residue is purified by flash chromatography to obtainethyl 2-amino-4-(4-bromophenyl)-thiophene-3-carboxylate. Ethyl2-amino-4-(4-bromophenyl)-thiophene-3-carboxylate (5 mmol) is treatedwith 4-fluorobenzoyl chloride (5 mmol) in THF (10 mL). The reaction isstirred at room temperature for 10 hours. The reaction is diluted with1N NaOH (10 mL) and ethyl acetate (15 mL). The layers are separated andthe organic layer is dried with anhydrous sodium sulfate, filtered andconcentrated. The residue is purified by flash chromatography to provideethyl 2-(4-fluorobenzamido)-4-(bromophenyl)thiophene-3-carboxylate.Ethyl 2-(fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylate ishydrolyzed under a variety of conditions (e.g. LiOH, water; or LiOH,H₂O₂, water; or NaOH, water) to provide2-(4-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid.

Example 2 2-(4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylicacid (Compound 2)

Ethyl cyanoacetate (5 mmol), and p-chloro-acetophenone (5 mmol) aredissolved in toluene (5 mL). Morpholine (5 mmol) is added followed byactivated molecular sieves (4A). The reaction is stirred at 80° C. for12 hours. The reaction is cooled to room temperature, filtered andconcentrated. The residue is taken up in toluene (5 mL), ethanol (5 mL)and sulfur is added (0.16 g; 5 mmol). The reaction mixture is heatedwith mixing at 70° C. for 12 hours. The reaction is cooled to roomtemperature and the solvents are evaporated. The residue is purified byHPLC to provide ethyl2-amino-4-(4-chlorophenyl)-thiophene-3-carboxylate. Ethyl2-amino-4-(4-chlorophenyl)-thiophene-3-carboxylate (5 mmol) is treatedwith 4-fluorobenzoyl chloride (5 mmol) in THF (10 mL). The reaction isstirred at room temperature for 10 hours. The reaction is diluted with1N NaOH (10 mL) and ethyl acetate (15 mL). The layers are separated, theorganic layer is dried with anhydrous sodium sulfate, filtered andconcentrated. The residue is purified by flash chromatography to provideethyl 2-(4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylate.Ethyl 2-(fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylate ishydrolyzed under a variety of conditions (e.g. LiOH, water; LiOH, H₂O₂,water; or NaOH, water) to provide2-(4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic acid.

Following the procedures outlined in Example 1 and Example 2, thefollowing compounds are prepared:

-   2-(4-fluorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic    acid;    2-(4-fluorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic    acid; 2-(4-fluorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic    acid;    2-(4-fluorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic    acid; 2-(4-fluorobenzamido)-4-(2-chlorophenyl)thiophene-3-carboxylic    acid;    2-(4-fluorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic    acid; 2-(4-fluorobenzamido)-4-(phenyl)thiophene-3-carboxylic acid;    2-(4-fluorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic acid;    2-(3-fluorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;    2-(4-chlorobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;    2-(4-iodobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic    acid;    2-(benzothien-2-ylamido)-4-(4-bromophenyl)thiophene-3-carboxylic    acid; 2-(3-methylbenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic    acid; 2-(4-bromobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic    acid; 2-(4-bromobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic    acid; 2-(4-bromobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic    acid;    2-(4-bromobenzamido)-4-(3,5-dichlorophenyl)thiophene-3-carboxylic    acid; 2-(4-bromobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic    acid;    2-(4-bromobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic    acid;    2-(4-bromobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic    acid;    2-(4-bromobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic    acid; 2-(4-bromobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic    acid; 2-(3-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic    acid;    2-(3-fluorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic    acid;    2-(3-fluorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic    acid; 2-(3-fluorobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic    acid; 2-(3-fluorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic    acid;    2-(3-fluorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic    acid; 2-(3-fluorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic    acid;    2-(3-fluorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic    acid; 2-(4-chlorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic    acid; 2-(4-chlorobenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic    acid;    2-(4-chlorobenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic    acid; 2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylic    acid;    2-(4-chlorobenzamido)-4-(3,4-dichlorophenyl)thiophene-3-carboxylic    acid;    2-(4-chlorobenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic    acid;    2-(4-chlorobenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic    acid; 2-(4-chlorobenzamido)-4-(phenyl)thiophene-3-carboxylic acid;    2-(4-chlorobenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;    2-(4-chlorobenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;    2-(benzothien-2-ylamido)-4-(4-chlorophenyl)thiophene-3-carboxylic    acid;    2-(benzothien-2-ylamido)-4-(4-methylphenyl)thiophene-3-carboxylic    acid;    2-(benzothien-2-ylamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic    acid;    2-(benzothien-2-ylamido)-4-(3-chlorophenyl)thiophene-3-carboxylic    acid;    2-(benzothien-2-ylamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic    acid;    2-(benzothien-2-ylamido)-4-(2-bromophenyl)thiophene-3-carboxylic    acid;    2-(benzothien-2-ylamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic    acid; 2-(3-methylbenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic    acid; 2-(3-methylbenzamido)-4-(4-methylphenyl)thiophene-3-carboxylic    acid; 2-(3-methylbenzamido)-4-(phenyl)thiophene-3-carboxylic acid;    2-(3-methylbenzamido)-4-(2-bromophenyl)thiophene-3-carboxylic acid;    2-(3-methylbenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylic acid;    2-(3-methylbenzamido)-4-(3-chlorophenyl)thiophene-3-carboxylic acid;    2-(3-methylbenzamido)-4-(4-trifluoromethylphenyl)thiophene-3-carboxylic    acid;    2-(3-methylbenzamido)-4-(2,4-dichlorophenyl)thiophene-3-carboxylic    acid;    2-(3-methylbenzamido)-4-(3,4-dimethylphenyl)thiophene-3-carboxylic    acid; Methyl    2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylate;    Ethyl    2-(4-chlorobenzamido)-4-(4-fluorophenyl)thiophene-3-carboxylate;    2-(4-iodobenzamido)-4-(4-bromophenyl)thiophene-3-carboxylic acid;    4-(2,4-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic    acid;    4-(3,4-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic    acid;    4-(3,5-dichlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic    acid;    4-(4-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic    acid;    4-(3-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic    acid;    4-(2-chlorophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(2-chloro-4-fluorobenzamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(3,4-difluorobenzamido)thiophene-3-carboxylic    acid;    2-(2-chloro-4-fluorobenzamido)-4-(4-chlorophenyl)thiophene-3-carboxylic    acid; 4-(4-bromophenyl)-2-(2-fluorobenzamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(3-fluoro-4-methoxybenzamido)thiophene-3-carboxylic    acid; 4-(4-chlorophenyl)-2-(4-methylbenzamido)thiophene-3-carboxylic    acid; 4-(4-bromophenyl)-2-(4-cyanobenzamido)thiophene-3-carboxylic    acid; 4-(4-chlorophenyl)-2-(4-ethylbenzamido)thiophene-3-carboxylic    acid;    4-(4-chlorophenyl)-2-(4-(trifluoromethyl)benzamido)thiophene-3-carboxylic    acid;    4-(4-chlorophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(3-(3-fluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-chlorophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(3-phenylpropanamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-chlorophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(3-(2,4-difluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-bromophenyl)-2-(3-(3,4-difluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-chlorophenyl)-2-(3-(4-fluorophenyl)propanamido)thiophene-3-carboxylic    acid;    4-(4-chlorophenyl)-2-(3-(3-chlorophenyl)propanamido)thiophene-3-carboxylic    acid; and    4-(4-chlorophenyl)-2-(3-(4-chlorophenyl)propanamido)thiophene-3-carboxylic    acid.

Biological Examples In Vitro Examples Example 3 In Vitro Screening forAgents that Modulate Intracellular Calcium Levels

Fluorescence-based assays were used for screening the compoundsdescribed herein, such as compounds of Formula (I) and Formula (II),which modulate intracellular calcium.

A. Assay Protocol

RBL-2H3 cells plated in 384-well plates were loaded for 45 min withFLUO-4-AM (2 μM final concentration) in HBSS. Cells were washed andplaced in a nominally Ca²⁺- and Mg²⁺-free Hanks solution. One minutelater, a test agent or vehicle was added. After a 15 minute incubationperiod, 1 μM thapsigargin (Tg) was added to inhibit the ER Ca²⁺ pump anddischarge intracellular Ca²⁺ stores. Fifteen minutes after addition ofTg, store-operated calcium entry was initiated by adding external Ca²⁺to a final concentration of 1.8 mM and the cells monitored for a further10-15 minutes. Calcium levels were monitored throughout the assay usinga FLIPR³⁸⁴ (Molecular Devices fluorimetric imaging plate reader for highthroughput screening).

In an alternative screening assay procedure, one minute after washingout the FLUO-4-AM, 1 μM Tg was added to the SH—SY5Y cells. Fifteenminutes after addition of Tg, test compound or vehicle was added,followed by another 15 minute incubation in Ca²⁺-free buffer.Store-operated calcium entry was then initiated by adding external Ca²⁺to a final concentration of 1.8 mM and the response monitored for afurther 10-15 minutes.

A similar screening assay procedure was used with HEK293 and RBL-2H3cells.

The screening assay can alternatively use external Ba²⁺ (finalconcentration of 10 mM) in place of external Ca²⁺. In this case,thapsigargin-induced store-operated Ba²⁺ entry serves as a surrogate forstore-operated Ca²⁺ entry.

B. Data Analysis

The kinetic data from the FLIPR³⁸⁴ were analyzed and then stored in arelational database (ActivityBase; IDBS). Ten quantitative parameterswere calculated that define various aspects of the store-operatedcalcium entry response. These parameters are as follows:

-   Mean Basal: basal fluorescence (relative fluorescence units, RFU)    readings averaged over 30 seconds prior to addition of Ca²⁺ to    initiate store-operated calcium entry.-   Up slope: linear regression of the increase in RFU from 2 to 30 sec    after addition of Ca²⁺.-   Up rate constant (Up K): the rate constant derived from first-order    association of RFUs from 2 seconds to peak response.-   Peak: the peak RFU (single point) achieved after addition of Ca²⁺.-   Time to peak: the time at which the peak RFU is achieved.-   Peak/Basal: the difference between peak and mean basal RFU.-   Decay slope: linear regression of the decrease in RFU from the peak    to the end of the measurement period.-   Decay rate constant (Decay K): the rate constant derived from    first-order decay of RFUs from the peak to the end of the    measurement period.-   Area under the curve (AUC): area under the curve from the addition    of Ca²⁺ to the end of the measurement period.

Combinations of these parameters were used to characterize the compoundsof Formula (I). Compounds were retested under identical conditions toconfirm their activity. Compounds with confirmed activity were thenanalyzed for concentration-dependent effects, and subsequently, thosecompounds displaying concentration-dependent effects were categorized ascompounds that modulate intracellular calcium.

TABLE A Representative compounds of Formula (I). Formula (I)

% Mean IC₅₀(μM) on Cmpd Inhibition Up Slope in RBL- no. R² —R¹ —R⁴ at 10μM 2H3 Cells 1 4-fluorophenyl —H 4-bromophenyl >70% 2.2 2 4-fluorophenyl—H 4-chlorophenyl — 2.3 3 4-fluorophenyl —H 3,4-dichlorophenyl >70% 3.34 4-fluorophenyl —H 4- >70% — trifluoromethylphenyl 11 3-fluorophenyl —H4-bromophenyl — 0.8 12 4-chlorophenyl —H 4-bromophenyl — 1.2 14benzothien-2-yl —H 4-bromophenyl >70% 2.4 15 3-methylphenyl —H4-bromophenyl — 2.6 16 4-bromophenyl —H 4-methylphenyl — 0.6 174-bromophenyl —H 4-chlorophenyl >70% 1 18 4-bromophenyl —H4-bromophenyl >70% 1.3 19 4-bromophenyl —H 3,5-dichlorophenyl >70% 1.620 4-bromophenyl —H 3-chlorophenyl <70% 4 21 4-bromophenyl —H3,4-dimethylphenyl >70% 4.6 25 3-fluorophenyl —H 4-chlorophenyl — 1.3 263-fluorophenyl —H 2,4-dichlorophenyl >70% 2.1 33 4-chlorophenyl —H4-chlorophenyl — 1 34 4-chlorophenyl —H 4-methylphenyl — 1 354-chlorophenyl —H 2,4-dichlorophenyl >70% 1.6 36 4-chlorophenyl —H4-fluorophenyl — 2.4 37 4-chlorophenyl —H 3,4-dichlorophenyl <70% 3 384-chlorophenyl —H 3,4-dimethylphenyl <70% 3.8 43 benzothien-2-yl —H4-chlorophenyl >70% 1.0 44 benzothien-2-yl —H 4-methylphenyl <70% 1.3 45benzothien-2-yl —H 2,4-dichlorophenyl >70% 2.4 46 benzothien-2-yl —H3-chlorophenyl >70% 10.7 50 3-methylphenyl —H 4-chlorophenyl — 1.2 513-methylphenyl —H 4-methylphenyl — 3 — = not determined.

Example 4 In Vitro Effects of Agents that Modulate Intracellular Calciumon Degranulation and Cytokine Release in RBL-2H3 Cells

To assess degranulation and cytokine release, RBL-2H3 cells were platedand stimulated with 20 nM thapsigargin/20 nM TPA for 20 hr in thepresence or absence of compound of Formula (I) or Formula (II). Mediawas collected and assayed for the release of β-hexosaminidase or for therelease of the inflammatory mediator cytokine TNF-α. Theβ-hexosaminidase enzymatic assay was performed by adding 200 μL 1 mMp-nitrophenyl-acetyl-glucosamide substrate (Sigma #N9376) in 0.05M NaCitrate (pH 4.5) to 50 μL of conditioned medium, incubating for 60 minat 37° C., then adding 500 μL 0.05M Na carbonate, 0.05M Na bicarbonatepH 10.5, mixing thoroughly, and reading the absorbance at 405 nm in aBioRad plate reader. The TNF-α release assay was performed using the RatTumor Necrosis Factor-a Ultrasensitive ELISA Kit from BioSource.

The results of these assays are shown in FIGS. 3 and 4.

Example 5 Modulation of Intracellular Calcium by a SOCE Inhibitor inSTIM1-Overexpressing Cells

Store-operated calcium entry is sensitive to the inhibitor2-aminoethoxydiphenyl borate (2-APB). To test whether the Ca²⁺ entrypathway activated by STIM1 overexpression is pharmacologically similarto endogenous SOCE, HEK[STIM1] cells were pre-incubated with increasingdoses of 2-APB and STIM1-dependent Ca²⁺ entry was measured.Thapsigargin-mediated store depletion of both HEK-Zeo control cells andHEK[STIM1] cells followed by readdition of external calcium resulted ininhibition by 2-APB with similar IC₅₀ values of 11.8 μM and 10.5 μM,respectively. Treatment of HEK[STIM1] cells with 2-APB and examiningcalcium entry in the absence of Tg-dependent store depletion resulted ina biphasic effect of 2-APB on calcium entry (FIG. 13). The calcium entryin this case was induced by pretreatment cells in a Ca²⁺-free buffer,and this Ca²⁺ entry was inhibited with an IC₅₀ value of 10.8 μM, similarto that reported for endogenous SOCE in HEK293 cells. However, at lowerconcentrations of 2-APB, calcium entry was potentiated. The ability toboth potentiate and inhibit calcium entry is a property of 2-APB thathas previously been shown to occur with the calcium release activatedcalcium (CRAC) channel.

Use of Compound 1 instead of 2-APB resulted in a concentration dependentinhibition of STIM1-dependent Ca²⁺ entry (see FIG. 13).

Thus, overexpression of STIM1 in HEK293 cells confers a CRAC-likeproperty to Ca²⁺ entry in HEK293 cells that is induced by exposing cellsto a Ca²⁺-free buffer without the requirement of store depletion bythapsigargin. Accordingly, assays to identify agents that modulateintracellular calcium can optionally be performed in cellsoverexpressing STIM1 in the absence of standard intracellular calciumdepletion protocols.

Example 6 In Vitro Effects of Agents that Modulate Intracellular Calciumon IL-2 Secretion from Jurkat T Cells.

To measure IL-2 secretion from Jurkat T cells, cells were plated in a 96well plate at a density of 1.5×10⁵ cells/well. Cells were stimulatedwith 2.5 μg/ml PHA lectin+80 nM TPA for 20 hr in the presence or absenceof a compound of Formula (I) or Formula (II). The medium was thencollected and analyzed for IL-2 levels by ELISA (BioSource) according tothe manufacturer's protocols.

The results of the assay are shown in FIGS. 7 and 10.

Example 6A In Vitro Effects of Agents on Cloned L-type Calcium Channelsin CHO Cells

Compound A stock solutions were prepared in dimethyl sulfoxide (DMSO)and stored frozen. Compound A concentrations were prepared fresh dailyby diluting stock solutions into an appropriate HEPES-bufferedphysiological saline solution. Each test compound formulation wassonicated at room temperatrue for at least 20 minutes to facilitatedissolution. A glass-lined 96-well compound plate was loaded with theappropriate amounts of test and control solutions, and placed in theplate well of PatchXpress®. The effects of five concentrations wereevaluated. Stock solutions of the positive control were prepared inbatches and frozen. The positive control concentration was preparedfresh daily by diluting stock solutions into HB-PS. The final DMSOconcentration was 0.3%.

Cells were maintained in tissue culture incubators per ChanTest SOP. CHOcells were stably transfected with the appropriate ion channel cDNAs.Cells were cultured in Ham's F-12 supplemented with 10% fetal bovineserum, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, andthe appropriate selection antibiotics. Cells in culture dishes werewashed twice with Hank's Balanced Salt Solution, treated with trypsinand re-suspended in the culture media before testing. Fiveconcentrations of each test compound A were applied at five minuteintervals to naive cells. Duration of exposure to each test compound wasapproximately five minutes.

In preparation for a recording session, intracellular solution wasloaded into the intracellular compartments of the planar electrode.After establishment of a whole-cell configuration, membrane currentswere recorded using dual-channel patch clamp amplifiers in thePatchXpress® system. Valid whole-cell recordings met the followingcriteria: 1. Membrane resistance (Rm)≧MΩ; 2. Leak current ≦25% channelcurrent. Onset and steady state block of hCay.1.2/β2/α2δ channels weremeasured using a stimulus voltage pattern consisting of a depolarizingtest pulse at 10 s intervals from a −80 mV holding potential. Testcompound concentrations were applied cumulatively in ascending orderwithout washout between applications. Peak current was measured duringthe step to 10 mV. Saturating concentration of nifedipine (10 μM) wasadded at the end of each experiment to block hCav1.2 current. Data werestored on the ChanTest computer network for analysis. Steady state wasdefined by the limiting constant rate of change with time. The steadystate before and after test compound application was used to calculatethe percentage of current inhibited at each concentration.

In Vivo Examples Example 7 Dose Responsive Effects of Compound 1,Compound 2, Cyclosporin A (CSA) or Rapamycin in Mouse Footpad DTH

Purpose: Determine dose responsive effects of test compound on mBSAinduced DTH response in foot pads when dosing is done during thesensitization as well as induction phase. Animals: 61 Male Swiss WebsterMice approx. 20-25 grams at start of study.

Materials: Methylated BSA (Sigma) Freund's complete adjuvant (Difco)plus supplemental M. tuberculosis H37 RA (Difco).

General Study Design:

Mice are anesthetized with Isoflurane and given intradermal antigeninjections of 0.1 ml at the base of the tail (D0, D07). Antigen isprepared by making a 4 mg/ml solution in sterile water. Equal volumes ofantigen and Freund's complete adjuvant to which 4 mg/ml MTB are added(sonicate for 5 minutes after adding MTB to oil), are emulsified by handmixing until a bead of this material holds its form when placed inwater. Treatment with test compound is initiated on day 0, qd (24 hrintervals) and continued through day 10 when challenge is done.

On day 10 animals are injected into the right hind footpad with 20 μl of10 mg/ml mBSA. Five unsensitized males are injected with mBSA into thefootpad. Twenty-four hours later (day 11) the right and left hind pawsare transected at the medial and lateral malleolus and weighed and theweight difference induced by injection of antigen is determined.

Statistical Analysis. Paw weights (mean±SE) for each group are analyzedfor differences using a Student's t test or ANOVA with Dunnett's posttest. Statistical significance is set at p<0.05.

TABLE 1 Treatment Groups Males Group N Treatment 10 ml/kg qd, po 1 5Normal controls (no sensitization) Inject mBSA into right only 2 8 DTH +Vehicle (70% PEG400/30% Water) 3 8 DTH + Test Compound (50 mg/kg, po,qd) 4 8 DTH + Test Compound (100 mg/kg, po, qd) 5 8 DTH + Test Compound(200 mg/kg, po, qd) 6 8 DTH + Test Compound (300 mg/kg, po, qd) 7 8DTH + CSA (100 mg/kg qd, ip) 8 8 DTH + Rapamycin (5 mg/kg qd, ip)

Protocol for the mouse DTH Study

The results for Compound 1 are presented in FIG. 12.

The results for Compound 2 are presented in FIG. 21.

Example 7A Pharmacokinetic Data of Compound 1 and Compound 2 in Rats

The bioavailability and plasma pharmacokinetic properties in rats ofCompound 1 administered orally in 70% PEG400/30% H₂O vehicle. Twotreatment groups, 1) an i.v. dose group at 2 mg/kg; and 2) an oral dosegroup at 10 mg/kg are administered to Male Sprague-Dawley rats (3 ratsper group), weighing approximately 250-300 gm. Up to 8 time points arecollected for each group. Typical time points are: predose, 15, 30minutes, 1, 2, 4, 8 and 24 hrs. Up to 300 μL of whole blood arecollected via jugular vein cannula at each time point. Whole blood iscollected into anticoagulant containing microcentrifuge tubes andcentrifuged at 5000 rpm in a microcentrifuge for 5 minutes before plasmais transferred to a clean microcentrifuge tube. The plasma samplesundergo bioanalytical analysis.

A similar procedure was used to obtain pharmacokinetic data for Compound2.

The results for Compound 1 are presented in FIG. 17.

The results for Compound 2 are presented in FIG. 21.

Example 8 Effect of Compound 1 in Rat Collagen Induced Arthritis (CIA)Model

Purpose: Determine efficacy of compound 1 administered by oral dosingqd, in inhibiting the inflammation, cartilage destruction and boneresorption of developing type II collagen arthritis in rats.

Animals: 44 Female Lewis rats (Charles River #7246950), weighing 125-150g at the start of the study. 40 rats are injected with collagen to get40 solid responders on days 10,11 for 4 groups of 10. Four nonimmunizedanimals serve as normal controls.

Materials: Compound 1 (sodium salt), PEG400 as liquid, Type II collagen,Freund's incomplete adjuvant, acetic acid. Compound 1 is prepared at aconcentration of up to 100 mg/ml in 70% PEG400/30% water. Collagen isprepared by making a 4 mg/ml solution in 0.01N Acetic acid. Equalvolumes of collagen and Freund's incomplete adjuvant, are emulsified byhand mixing until a bead of this material holds its form when placed inwater.

General Study Design: Animals (10 rats/group for arthritis, 4 rats/groupfor normal control).

Animals in groups 2-5 are anesthetized with isoflurane and givencollagen injections (DO); each animal gets 300 μl of the mixture spreadover 3 subcutaneous sites on the back. On Day 6 (D6) the animals areanesthetized again and given a second collagen injection, as before.

Oral dosing of Compound 1 at 24 hour intervals (qd) is initiated on Day0 using a dose volume of 5 ml/kg for oral solutions. Rats are weighed onDays 0, 3, 6, and 9-17 of arthritis, and caliper measurements of anklestaken every day beginning on Day 9. Final body weights are taken on Day17 of arthritis. On Day 17, all animals are anesthetized for terminalblood draw and then euthanized. Subsequently, hind paws and knees areremoved, the hind paws are weighed and then (with knees) placed informalin for processing for microscopy. Livers, spleen and thymus andkidneys are also removed, trimmed of extraneous tissue and weighed.Kidneys are retained in formalin for histopathology.

Sampling will occur over 1 day and involves groups 2-5 with samplesretained from all groups. This results in all animals being treatedsimilarly and is important for clinical parameters and final liverweights.

The results of the study are shown in FIG. 11.

Example 9 Effect of Compound 1 on DNBS-Induced Colitis in Rats

Procedure: Male Wistar rats weighing 200±20 g are fasted for 24 hoursprior to use. Distal colitis is induced by intra-colonic instillation ofDNBS (2,4-dinotrobenzene sulfonic acid, 20 mg in 0.5 ml ethanol 30%)with a catheter of 12 cm in length, followed by gentle injection of air(2 ml) through the catheter to ensure that the solution remain in thecolon. The animals are divided into groups of 5 each. Test substance andvehicle are administered either daily or twice daily by appropriateroute of administration 24 hour and 1 hour before DNBS instillation andthen for 6 consecutive days thereafter. One normal control group istreated with 0.9% NaCl alone without DNBS challenge. The animals aresacrificed 12 hours after the final bid dose and 24 hours after thefinal daily dose and the colon is removed and weighed. During theexperiment, body weight, fecal occult blood and stool consistency aremonitored daily. Furthermore, when the abdominal cavity is opened beforeremoval of the colon, adhesions between the colon and other organs arenoted as is the presence of colonic ulceration after removal andweighing of each colon (a macroscopic damage score is recorded accordingto established score criteria). The colon-to-body weight ratio iscalculated according to the formula: Colon (g)/BW×100. The “Net”increase in ratio of Vehicle-control+DNBS group relative toVehicle-control group is used as a base for comparison with individualtreated groups and expressed as “Dec. (%)” (percent decrease). A 30% ormore (≧30%) reduction in colon-to-body weight ratio, relative to thevehicle treated control group, is considered significant.

Sulfasalazine is used the standard test agent. (Hogaboam C M, et al., Anorally active non-selective endothelin receptor antagonist, bosentan,markedly reduces injury in a rat model of colitis. Eur J Pharmacol. 309:261-269, 1996; Yue G, et al., The 21-aminosteroid tirilazid mesylate canameliorate inflammatory bowel disease in rats. J Pharmacol Exp Ther.276: 265-270, 1996.)

The results of the study are shown in FIG. 20.

Example 10 Phase II Clinical Trial of the Safety and Efficacy ofCompounds of Formula (I), (II), (IIA), or (IIB) in Patients with ActiveRheumatoid Arthritis.

The purpose of this phase II trial is to investigate the safety,tolerability, PK, PD, and efficacy of single and repeat intravenousinfusions of a compound of Formula (I), (II), (IIA), or (IIB) inpatients with active rheumatoid arthritis.

-   Patients: Eligible subjects will be men and women between the ages    of 18 and 75

Criteria:

Inclusion Criteria:

-   -   All subjects must use acceptable contraception to ensure that no        pregnancies occur during the course of the study and for at        least 12 weeks after dosing for males and for 32 weeks after        dosing for females;    -   Body mass index within the range 18.5-35 kg/m² inclusive, in        addition to a weight range of 55-95 kg;    -   The subject must be capable of giving informed consent and can        comply with the study requirements and timetable;    -   The subject must have a diagnosis of RA according to the revised        1987 criteria of the American College of Rheumatology (ACR);    -   The subject must have a DAS28 disease activity score of greater        than 4.2 at screening and pre-dose;    -   The subject must have a CRP serum level of >/0.5 mg/dl or an ESR        level 28 mm/hour at screening and pre-dose;    -   The subject has NOT received any biological therapy in the past,        including biologicals for the treatment of rheumatoid arthritis;    -   The subject must have liver function tests including alanine        transaminase (ALT) and aspartate transaminase (AST) within 1.5        times the upper limit of normal (ULN) and alkaline phosphatase        (ALP) within 3 times ULN at screening. The patient must also        have total bilirubin within the ULN at screening;    -   The subject must have received at least 3 months of methotrexate        and must be on a stable dose of methotrexate (up to 25 mg/week)        for at least 8 weeks prior to screening and be willing to remain        on this dose throughout the study;    -   If sulfasalazine is being taken in addition to methotrexate, the        subject must be on a stable dose for at least 4 weeks prior to        screening and be willing to remain on this dose throughout the        study;    -   If hydroxychloroquine or chloroquine is being taken in addition        to methotrexate, the subject must be on a stable dose for at        least 3 months prior to screening and be willing to remain on        this dose throughout the study;    -   Those subjects on other oral anti-rheumatic therapies, which may        include Non Steroidal Anti

Inflammatory Drugs (NSAIDs), COX-2 inhibitors, oral glucocorticoids e.g.prednisolone (˜10mg/day) must be on stable dosing regimens for at least4 weeks prior to screening and be willing to remain on this regimethroughout the study. Subjects receiving intramuscular glucocorticoidse.g methylprednisolone (˜120 mg/month) must be on a stable dosingregimen for at least 3 months prior to screening and be willing toremain on this regimen throughout the study;

-   -   The subject must be on a stable dose of folate supplements (5        mg/week) for at least 4 weeks prior.

Exclusion Criteria:

-   -   Any clinically relevant abnormality identified on the screening        medical assessment, laboratory examination (e.g. haematology        parameter outside the normal limits), or ECG (12 Lead or        Holter);    -   The subject has a positive Hepatitis B surface antigen or        Hepatitis C antibody result at screening;    -   The subject has a history of elevated liver function tests on        more than one occasion (ALT, AST and ALP>3×Upper Limit of Normal        (ULN); total bilirubin>1.5×ULN) in the past 6 months;    -   Previous exposure or past infection caused by Mycobacterium        tuberculosis;    -   The subject has an acute infection;    -   The subject has a history of repeated, chronic or opportunistic        infections that, in the opinion of the investigator and/or GSK        medical monitor, places the subject at an unacceptable risk as a        participant in this trial;    -   The subject has a history of malignancy, except for surgically        cured basal cell carcinoma or females with cured cervical        carcinoma (>2 yrs prior);    -   The subject has a history of human immunodeficiency virus (HIV)        or other immunodeficiency disease;    -   The subject whose calculated creatinine clearance is less than        50 ml/min;    -   The subject has significant cardiac, pulmonary, metabolic,        renal, hepatic or gastrointestinal conditions that, in the        opinion of the investigator and/or GSK medical monitor, places        the subject at an unacceptable risk as a participant in this        trial;    -   The subject has taken cyclosporine, leflonomide, cyclophophamide        or azathioprine within 1 month of screening. Subjects that have        taken cyclosporine, leflonomide, cyclophophamide or azathioprine        in the past must have recovered from all drug related adverse        events;    -   The subject has taken gold salts or d-penicillamine within 1        month prior to screening.

Subjects that have taken gold salts or d-penicillamine in the past musthave recovered from all drug related adverse events;

-   -   The subject has received intra-articular glucocorticoids within        1 month of screening;    -   Recent history of bleeding disorders, anaemia, peptic ulcer        disease, haematemesis or gastrointestinal bleeding;    -   Subjects with a history of haematological disease or acquired        platelet disorders, including drug-induced thrombocytopaenia,        acute idiopathic thrombocytopaenia or von Willebrand's disease;    -   Subjects with a known risk of intra-cranial haemorrhage        including Central Nervous System (CNS) surgery within the last        12 months, arterial vascular malformations, aneurysms,        significant closed head trauma within 6 months or any other        incident the investigator and/or medical monitor considers to be        relevant;    -   The subject has Hb<10 g/deciliter (dL) and platelet        count<150×109/Liter (L);    -   Donation of blood in excess of 500 ml within a 56 day period        prior to dosing;    -   An unwillingness of male subjects to abstain from sexual        intercourse with pregnant or lactating women; or an        unwillingness of the male subject to use a condom with        spermicide in addition to having their female partner use        another form of contraception such as an interuterine device        (IUD), diaphragm with spermicide, oral contraceptives,        injectable progesterone, subdermal implants of levonorgestrel or        a tubal ligation if the woman could become pregnant for at least        12 weeks after dosing;    -   An unwillingness of female subject of child bearing potential to        use adequate contraception, as defined in the study restriction        section. If necessary, women of non-child bearing potential        (i.e. post-menopausal or surgically sterile e.g. tubal ligation        or hysterectomy or bilateral oophorectomy) will be confirmed.        Postmenopausal status will be confirmed by serum follicle        stimulating hormone (FSH) and oestradiol concentrations at        screening. Surgical sterility will be defined as females who        have had a documented hysterectomy, tubal ligation or bilateral        oophorectomy;    -   The subject has a history of use of drugs of abuse within 12        months prior to screening;    -   History of regular alcohol consumption exceeding average weekly        intake of greater than 21 units or an average daily intake of        greater than 3 units (males) or an average weekly intake of        greater than 14 units or an average daily intake of greater than        2 units (females). Subjects who regularly consume more than 12        units of alcohol in a 24 h period will also be excluded. 1 unit        is equivalent to a half-pint (220 ml) of beer/lager or 1 (25 ml)        measure of spirits or 1 glass (125 ml) of wine;    -   Positive pregnancy test or lactating at screening;    -   Participation in a trial with any investigational drug within 3        months or 5 half-lives (whichever is longer) before.

-   Study Design: This is a randomized, double-blinded,    placebo-controlled adaptive, dose finding study to investigate the    safety, tolerability, PK, PD and efficacy of single and repeat    intravenous infusions of a compound of Formula (I), (II), (IIA), or    (IIB) in patients with active rheumatoid arthritis. The study is    divided into 2 parts: Part A is an adaptive, dose finding phase    which will provide safety, tolerability, PK and PD on single    intravenous infusions. Part B is a repeat dose phase which will    provide safety, tolerability, PK, PD and efficacy following repeat    intravenous infusions of a selected dose level.

Primary Outcome Measures:

-   -   Safety and Tolerability following single ascending doses of a        compound of Formula (I), (II), (IIA), or (IIB) at 1 month and        following 3 repeat doses of a compound of Formula (I), (II),        (IIA), or (IIB) at 3 months. Clinical Efficacy (DAS28 score) of        a compound of Formula (I), (II), (IIA), or (IIB) at 1 month

Secondary Outcome Measures:

-   -   Weighted mean DAS28 after single and repeat intravenous doses    -   Plasma PK parameters of a compound of Formula (I), (II), (IIA),        or (IIB) after single and repeat intravenous doses including        free, and bound a compound of Formula (I), (II), (IIA), or (IIB)        (serum) concentrations, AUC_((0-∞)), C_(max), clearance, volume        of distribution and accumulation ratio    -   DAS28 and EULAR response criteria after single and repeat        intravenous doses    -   ACR20/ACR50/ACR70 response after single and repeat intravenous        doses    -   Number of swollen joints assessed using 28-joint counts    -   Number of tender/painful joints assessed using 28-joint counts    -   Subject's pain assessment    -   Physician's global assessment of arthritis condition    -   Patients' global assessment of arthritis condition    -   Functional disability index (Health Assessment Questionnaire)    -   C-reactive Protein (CRP)    -   ESR    -   Global Fatigue Index    -   HAQ disability index    -   Pharmacodynamic biomarkers after single and repeat intravenous        doses    -   Characteristic AUC₅₀ and EC₅₀ for clinical endpoint changes with        plasma exposure model, as assessed by sigmoid E. and indirect        response PK/PD models.    -   Immunogenicity (Human anti-compound of Formula (I), (II), (IIA),        or (IIB) antibodies)

Example 11 Phase II Clinical Trial of the Safety and Efficacy ofCompounds of Formula (I), (II), (IIA), or (IIB) in Patients with Severe,Recalcitrant, Plaque-Type Psoriasis.

The purpose of this phase II trial is to investigate the safety,efficacy, and tolerability of a compound of Formula (I), (II), (IIA), or(IIB) in patients with severe, recalcitrant, plaque-type psoriasis.

-   Patients: Eligible subjects will be men and women between the ages    of 18 and 75.

Criteria:

Inclusion Criteria:

-   -   The patient has severe, recalcitrant, plaque-type psoriasis and        has failed at least 1 systemic therapy (for the purposes of this        study psoralen with ultraviolet light A is considered to be a        systemic therapy);    -   The patient has psoriatic involvement of at least 10% of BSA;    -   The patient has a PSGA score of 4 or greater;    -   The patient, if a woman, is surgically sterile or 2 years        postmenopausal, or if of childbearing potential is currently        using a medically accepted method of contraception, and agrees        to continue use of this method for the duration of the study        (and for 30 days after participation in the study). Acceptable        methods of contraception include: abstinence, steroidal        contraceptive (oral, transdermal, implanted, or injected) in        conjunction with a barrier method, or intrauterine device (IUD);    -   The patient, if a main, is surgically sterile, or if capable of        producing offspring, is currently using an approved method of        birth control, and agrees to continued use of this method for        the duration of the study (and for 60 days after taking the last        dose of a compound of Formula (I), (II), (IIA), or (IIB) because        of the possible effects on spermatogenesis);    -   The patient must be willing and able to comply with study        procedures and restrictions and willing to return to the clinic        for the follow-up evaluation as specified in this protocol.

Exclusion Criteria:

-   -   The patient has received treatment with systemic psoriasis        treatments (specifically, retinoids, methotrexate, cyclosporine        A, etanercept, efalizumab, other biological agents or other        immunomodulators) within 4 weeks, or UV based therapy within 2        weeks, or alefacept within 6 weeks of the planned 1st day of        study treatment;    -   The patient has received treatment with potent CYP3A4 inhibitors        including cyclosporine, clotrimazole, fluconazole, itraconazole,        ketoconazole, voriconazole, erythromycin, clarithromycin, and        troleandomycin, human immunodeficiency virus (HIV) protease        inhibitors, or nefazodone within 1 week (7 days) of the planned        1st day of study treatment;    -   The patient is currently receiving warfarin;    -   The patient has hypersensitivity to a compound of Formula (I),        (II), (IIA), or (IIB) or any component of a compound of Formula        (I), (II), (IIA), or (IIB);    -   The patient has one or more of the following serum chemistry        values as determined at the screening visit (visit 1):    -   bilirubin levels greater than 2 times the upper limit of normal        (ULN);    -   ALT or AST levels greater than 2 times the ULN;    -   serum creatinine levels or more than 2 mg/dL;    -   The patient requires current treatment for HIV with protease        inhibitors;    -   The patient is taking medication for a clinical diagnosis of        gastrointestinal ulceration or has experienced melena or        hematoemesis in the previous 3 weeks;    -   The patient is a woman who is pregnant or lactating;    -   The patient has received treatment with an investigation drug        within 4 weeks of the planned 1st day of study treatment.

-   Study Design: This is an exploratory, open-label, nonrandomized,    dose-escalation study of the efficacy, safety, and tolerability of a    compound of Formula (I), (II), (IIA), or (IIB) in patients with    severe, recalcitrant, plaque-type psoriasis.

Example 12 Phase II Clinical Trial of the Safety and Efficacy ofCompounds of Formula (I), (II), (IIA), or (IIB) for Prophylaxis of AcuteRejection after Renal Transplantation

The standard immunosuppressive treatment after renal transplantation isa combination of tacrolimus, mycophenolate mofetil, and prednisolone.With this regimen the incidence of acute rejection within the first sixmonths after transplantation can drop to about 20%. The main challengeat present remains to improve long-term outcome by preventing chronicallograft nephropathy (CAN). Since acute rejection is a strong predictorof CAN, a further decrease in the incidence of acute rejection canimprove the long-term graft survival. The purpose of this phase IIclinical trial is to investigate the effectiveness and safety of acompound of Formula (I), (II), (IIA), or (IIB) for prophylaxis of acuterejection after renal transplantation.

-   Patients: Eligible subjects will be men and women ages 18 and older

Criteria:

Inclusion Criteria:

-   -   Renal transplant recipients;    -   Signed, dated, and witnessed IRB approved informed consent;

Exclusion Criteria:

-   -   Pregnancy;    -   Living donor, who is HLA identical;    -   Hemolytic uremic syndrome as original kidney disease;    -   Focal segmental glomerulosclerosis that had recurred in a        previous graft;    -   More than two previously failed grafts and/or PRA>85%;    -   Diabetes mellitus that is currently not treated with insulin;    -   Total white blood cell count<3,000/mm3 or platelet        count<75,000/mm3;    -   Active infection with hepatitis B, hepatitis C, or HIV;    -   History of tuberculosis.

-   Study Design: This is a randomized, double blind, placebo controlled    intervention study on the efficacy and safety of the prophylatic use    of a compound of Formula (I), (II), (IIA), or (IIB). One group will    receive a single dose of a compound of Formula (I), (II), (IIA), or    (IIB) intravenously at the time of transplantation, and the other    group receives a placebo infusion.

Primary Outcome:

-   -   To determine the incidence and severity of biopsy-confirmed        acute rejection within the first six months after        transplantation

Secondary Outcomes:

-   -   Renal function as estimated by the endogenous creatinine        clearance at 6 months    -   Occurrence of chronic allograft nephropathy at 6 months    -   Cumulative incidence of infections and malignancies at 6 months    -   Medical costs during the first 6 months after transplantation    -   Patient and graft survival

Example 13 Phase II Clinical Trial of the Safety and Tolerability of aCompound of Formula (I), (II), (IIA), or (IIB) in Patients with ActiveUlcerative Colitis (UC)

The purpose of this phase II trial is to investigate the safety,tolerability of a compound of Formula (I), (II), (IIA), or (IIB) regimenin patients with active ulcerative colitis.

-   Patients: Eligible subjects will be men and women aged 18 and older

Criteria:

Inclusion Criteria:

-   -   Active UC on 5-ASA therapy and also treated with 6-MP and/or        corticosteroids or who have previously been treated with AZA,        6-MP or corticosteroids and could not tolerate them;    -   Mayo score of 6 to 10 points with moderate to severe disease on        endoscopy (Mayo score of at least 2) performed ≦14 days of study        drug administration;    -   Subjects on the following medications may be enrolled into the        study if the medications were according to the following        schedules prior to study drug administration and if no changes        are anticipated during the study;        -   prednisolone ≦20 mg daily (or equivalent) (dose must be            stable for at least 2 weeks prior to study drug            administration);        -   5-ASA (dose must be stable for at least 4 weeks prior to            study drug administration);        -   AZA or 6-MP (dose must be stable for at least 3 months prior            to study drug administration);        -   Rectal steroids or 5-ASA (must have been stable for at least            4 weeks prior to study drug);    -   Subjects using rectal medications must have visible disease on        sigmoidoscopy at ≧20 cm;    -   Screening laboratory values must meet certain criteria:        -   Women must be postmenopausal (>12 months without menses) or            surgically sterile (e.g., by hysterectomy and/or bilateral            oophorectomy) or must be using effective contraception            (e.g., oral contraceptives, intrauterine device (IUD),            double barrier method of condom and spermicidal) for at            least 4 weeks prior to study drug administration and agree            to continue contraception for the duration of their            participation in the study; and        -   Sexually active male subjects must use a barrier method of            contraception during the duration of the study

Exclusion Criteria:

-   -   Anti-TNF therapy within 8 weeks before study drug        administration;    -   Any experimental therapy more therapy ≦4 weeks before study drug        administration;    -   Prior treatment with any monoclonal antibody or        immunoglobulin-based fusion proteins ≦8 weeks prior to study        treatment;    -   Presence of Cushing's syndrome;    -   Toxic megacolon or fulminant disease likely to require        colectomy;    -   Contraindication to colonoscopy or sigmoidoscopy;    -   Primary or secondary immunodeficiency;    -   Autoimmune disease besides UC, with the exceptions of Sjogren's        syndrome or hypothyroidism;    -   History of malignancy, excluding adequately treated and cured        basal or squamous cell of the skin, or cervical carcinoma in        situ;    -   Major psychiatric disease (subjects with stable depression        receiving appropriate management will be permitted in the        study);    -   Evidence of acute or chronic infection as evidenced by:    -   stool culture positive for pathogens and/or Clostridium        difficile toxin;    -   findings on Screening chest radiography such as pulmonary        infiltrate(s) or adenopathy;    -   current treatment for tuberculosis infection, clinical or        radiological evidence of active TB, or for subjects in North        America, a positive PPD without prior prophylaxis;    -   Herpes zoster ≦3 months prior to study drug administration;    -   active infectious disease requiring i.v. antibiotics within 4        weeks prior to study treatment or oral antibiotics at the time        of enrollment;    -   HIV or AIDS;    -   positive tests for HBV, or HCV indicating active or chronic        infection;    -   Clinically significant cardiac disease requiring medication,        unstable angina, myocardial within 6 months, or congestive heart        failure;    -   Arrhythmia requiring active therapy, with the exception of        clinically insignificant or minor conduction abnormalities;    -   History of cerebrovascular disease requiring        medication/treatment;    -   Anticoagulation therapy or a known bleeding disorder;    -   Seizure disorder requiring active therapy;    -   Known drug or alcohol abuse;    -   Pregnant or nursing;    -   Any underlying medical condition that in the Principal        Investigator's opinion will make the study drug hazardous to the        subject or would obscure the interpretation of treatment        efficacy or safety; or    -   Inability or unwillingness to return for Follow-up visits and        comply with study protocol

Primary Outcome Measures:

-   -   Change in Mayo score at Day 57 compared with Screening

Secondary Outcome Measures:

-   -   Remission rate

Study Design:

This is a phase II, double-blind, placebo-controilled, randomized,multi-dose study of a compound of Formula (I), (II), (IIA), or (IIB) insubjects with active UC experiencing flare. All subjects will haveactive disease while on a 5-ASA containing medication and are either onstable doses of corticosteroids and/or azathioprine or 6-mercaptopurine,or who have previously been on these medications but could not toleratethem. Flare is defined as a Mayo score of 6 to 10 with moderate tosevere disease activity on endoscopy (Mayo endoscopic subscore of atleast 2) within 2 weeks of receiving study drug administration. Doses ofpermitted concomitant medications (corticosteroids, azathioprine (AZA),6-mercaptopurine (6-MP), and 5-aminosalicylates (5-ASA) containingcompounds) should remain constant during the course of the study.Subjects will be randomized to receive placebo or a compound of Formula(I), (II), (IIA), or (IIB) intravenously on Days 1, 15, 29, and 43. Allsubjects will be seen in the clinic at regular intervals up to Day 85for safety, efficacy, pharmacokinetic, and/or pharmacodynamicassessments. All subjects will be contacted 70 days after the last doseof study drug. Assessment of safety will be determined by vital signmeasurements, clinical laboratory tests, physical examinations,immunogenicity assessments, chest x-ray, electrocardiograms, and theincidence and severity of treatment emergent adverse events. The primaryclinical assessment of activity will be determined by the change in Mayoscore at Day 57 compared with Screening. Secondary endpoints includedetermination of remission rate by the mayo score at Day 57, evaluationof mucosal healing and change from baseline in the IBDQ score.

Example 14 Phase II Clinical Trial of the Safety and Efficacy ofCompounds of Formula (I), (II), (IIA), or (IIB) in Patients withMultiple Sclerosis

The purpose of this phase II trial is to investigate the safety,efficacy and tolerability of a compound of Formula (I), (II), (IIA), or(IIB) in patients with Relapsing-Remitting Multiple Schlerosis.

-   Patients: Eligible subjects will be men and women between the ages    of 18 and 65.

Criteria:

Inclusion Criteria:

-   -   Have a definite diagnosis of Relapsing remitting Multiple        Sclerosis    -   Have a history of at least 1 of the following:a. A minimum of 2        relapses of MS within the previous 2 years but not within the        1-month period prior to screening. b. A relapse of MS within the        previous 6 months but not within the 1-monthperiod prior to        screening

Exclusion Criteria:

-   -   Have a CNS disease (eg, CNS lymphoma, systemic lupus        erythematous)    -   Have significant bulbar involvement of MS or other neurologic        deficits    -   Have a decubitus ulcer    -   Have received immunomodulatory therapies within 3 months of        screening

Primary Outcome Measures:

-   -   The cumulative number of newly Gd-enhancing T1-weighted lesions        on cranial MRIs through week 23

Secondary Outcome Measures:

-   -   The total number of relapses of MS through week 23; change from        baseline in Expanded Disability Status Scale (EDSS) score at        week 23

Study Design: This is a phase II, double-bline, placebo-controlled,randomized, dose-ranging study of multiple subcutaneous injections of acompound of Formula (I), (II), (IIA), or (IIB) in patients withrelapsing-remitting multiple sclerosis. Patients will receivesubcutaneous injections of a compound of Formula (I), (II), (IIA), or(IIB) or placebo at weeks 0, 1, 2, 3, 7, 11, 15, and 19 or 100.

Pharmaceutical Compositions

Parenteral Composition

To prepare a parenteral pharmaceutical composition suitable foradministration by injection, 100 mg of a compound of Formula (I), (II),(IIA) or (IIB) is dissolved in DMSO and then mixed with 10 mL of 0.9%sterile saline. The mixture is incorporated into a dosage unit formsuitable for administration by injection.

In another embodiment, the following ingredients are mixed to form aninjectable formulation:

Ingredient Amount Compound of Formula (I), (II), (IIA), or (IIB) 1.2 gsodium acetate buffer solution (0.4M)  2.0 mL HCl (1N) or NaOH (1M) q.s.to suitable pH water (distilled, sterile) q.s. to 20 mL

All of the above ingredients, except water, are combined and stirred andif necessary, with slight heating if necessary. A sufficient quantity ofwater is then added.

Oral Composition

To prepare a pharmaceutical composition for oral delivery, 100 mg of acompound of Formula (I), (II), (IIA) or (IIB) is mixed with 750 mg ofstarch. The mixture is incorporated into an oral dosage unit, such as ahard gelatin capsule, which is suitable for oral administration.

In another embodiment, the following ingredients are mixed intimatelyand pressed into single scored tablets.

Ingredient Quantity per tablet, mg compound of Formula (I), (II), (IIA),or (IIB) 200 Cornstarch 50 croscarmellose sodium 25 Lactose 120magnesium stearate 5

In yet another embodiment, the following ingredients are mixedintimately and loaded into a hard-shell gelatin capsule.

Ingredient Quantity per tablet, mg compound of Formula (I), (II), (IIA),or (IIB) 200 lactose, spray-dried 148 magnesium stearate 2

In yet another embodiment, the following ingredients are mixed to form asolution/suspension for oral administration:

Ingredient 1. Amount  2. Compound of Formula (I), (II), (IIA), or (IIB) 4. 1 g  3. Anhydrous Sodium Carbonate  5. 0.1 g  6. Ethanol (200proof), USP  7. 10 mL  8. Purified Water, USP  9. 90 mL 10. Aspartame11. 0.003 g

Sublingual (Hard Lozenge) Composition

To prepare a pharmaceutical composition for buccal delivery, such as ahard lozenge, mix 100 mg of a compound of Formula (I), (II), (IIA) or(IIB) with 420 mg of powdered sugar mixed with 1.6 mL of light cornsyrup, 2.4 mL distilled water, and 0.42 mL mint extract. The mixture isgently blended and poured into a mold to form a lozenge suitable forbuccal administration.

Inhalation Composition

To prepare a pharmaceutical composition for inhalation delivery, 20 mgof a compound of Formula (I), (II), (IIA) or (IIB) is mixed with 50 mgof anhydrous citric acid and 100 mL of 0.9% sodium chloride solution.The mixture is incorporated into an inhalation delivery unit, such as anebulizer, which is suitable for inhalation administration.

Rectal Gel Composition

To prepare a pharmaceutical composition for rectal delivery, 100 mg of acompound of Formula (I), (II), (IIA) or (IIB) is mixed with 2.5 g ofmethylcelluose (1500 mPa), 100 mg of methylparapen, 5 g of glycerin and100 mL of purified water. The resulting gel mixture is then incorporatedinto rectal delivery units, such as syringes, which are suitable forrectal administration.

Suppository Formulation

A suppository of total weight 2.5 g is prepared by mixing a compound ofFormula (I), (II), (IIA) or (IIB) with Witepsol™ H-15 (triglycerides ofsaturated vegetable fatty acid; Riches-Nelson, Inc., New York), and hasthe following composition:

Quantity per Ingredient suppository, mg compound of Formula (I), (II),(IIA), or (IIB) 500 Witepsol ® H-15 balance

Topical Gel Composition

To prepare a pharmaceutical topical gel composition, 100 mg of acompound of Formula (I), (II), (IIA) or (IIB) is mixed with 1.75 g ofhydroxypropyl cellulose, 10 mL of propylene glycol, 10 mL of isopropylmyristate and 100 mL of purified alcohol USP. The resulting gel mixtureis then incorporated into containers, such as tubes, which are suitablefor topical administration.

Ophthalmic Solution Composition

To prepare a pharmaceutical opthalmic solution composition, 100 mg of acompound of Formula (I), (II), (IIA) or (IIB) is mixed with 0.9 g ofNaCl in 100 mL of purified water and filterd using a 0.2 micron filter.The resulting isotonic solution is then incorporated into ophthalmicdelivery units, such as eye drop containers, which are suitable forophthalmic administration.

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the purview of disclosureand scope of the appended claims.

1.-5. (canceled)
 6. A method of treating a disease, disorder orcondition in a mammal that would benefit from inhibition of storeoperated calcium channel activity comprising administering to the mammala compound of Formula (I), or pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof:

wherein: R¹ is hydrogen, C₁-C₆alkyl, C₁-C₆haloalkyl, or benzyl; R² isaryl, benzothienyl, benzofuranyl, or —CH₂CH₂-phenyl; wherein R² isoptionally substituted with 1 or 2 substituents independently selectedfrom F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OCF₃, —OR⁸, C₁-C₆alkyl,C₃-C₆cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, tetrazolyl,C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸, —S(═O)₂N(R⁹)₂, —C(═O)CF₃,—C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁸, —N(R⁹)₂, —N(R⁹)C(═O)R⁸, —CO₂R⁹,—C(═O)R⁸, —OC(═O)R⁸, —CON(R⁹)₂, —SR⁸, —S(═O)R⁸, and —S(═O)₂R⁸ R⁴ is anaryl, wherein the aryl is optionally substituted with 1 or 2substituents selected from F, Cl, Br, I, —CN, —NO₂, —CF₃, —OH, —OR⁸,—OCF₃, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆fiuoroalkyl, C₁-C₆heteroalkyl,C₁-C₆haloalkyl, tetrazolyl, C₂-C₆heterocycloalkyl, phenyl, —NHS(═O)₂R⁸,S(═O)₂N(R⁹)₂, —C(═O)CF₃, —C(═O)NHS(═O)₂R⁸, —S(═O)₂NHC(═O)R⁹, N(R⁹)₂,—N(R⁹)C(═O)R⁸, —CO₂R⁹, —C(═O)R⁸, —OC(═O)R⁸, —C(═O)N(R⁹)₂, -SR⁸,—S(=0)R⁸, and —S(═O)₂R⁸; each R⁸ is independently selected fromC₁-C₆alkyl, C₁-C₆haloalkyl, C₃-C₈cycloalkyl, phenyl, and benzyl; each R⁹is independently selected from H, C₁-C₆alkyl, C₁-C₆haloalkyl,C₃-C₈cycloalkyl, phenyl, and benzyl.
 7. The method of claim 6 wherein:the disease, disorder or condition in a mammal is selected fromdiseases/disorders involving inflammation, glomerulonephritis, uveitis,hepatic diseases or disorders, renal diseases or disorders, chronicobstructive pulmonary disease, rheumatoid arthritis, psoriasis,inflammatory bowel disease, vasculitis, dermatitis, osteoarthritis,inflammatory muscle disease, allergic rhinitis, vaginitis, interstitialcystitis, scleroderma, osteoporosis, eczema, organ transplant rejection,allogeneic or xenogeneic transplantation, graft rejection,graft-versus-host disease, lupus erythematosus, type I diabetes,pulmonary fibrosis, dermatomyositis, thyroiditis, myasthenia gravis,autoimmune hemolytic anemia, cystic fibrosis, chronic relapsinghepatitis, primary biliary cirrhosis, allergic conjunctivitis, hepatitisand atopic dermatitis, asthma, multiple sclerosis, Sjogren's syndrome,and autoimmune diseases or disorders.
 8. The method of claim 7 whereinthe disease, disorder or condition is rheumatoid arthritis. 9.(canceled)
 10. The method of claim 7 wherein the disease, disorder, orcondition is inflammatory bowel disease.
 11. (canceled)
 12. The methodof claim 7 wherein the disease, disorder, or condition is multiplesclerosis. 13.-24. (canceled)
 25. The method of claim 6 wherein R¹ ofthe compound is hydrogen.
 26. The method of claim 6 wherein R² of thecompound is benzothienyl optionally substituted with 1 or 2 substituentsselected from F, Cl, Br, I, OH, CH₃, CF₃, and CN.
 27. The method ofclaim 26 wherein the benzothienyl is attached at the 3-position.
 28. Themethod of claim 26 wherein the benzothienyl is attached at the5-position.
 29. The method of claim 6 wherein R⁴ of the compound is anaryl group.
 30. The method of claim 29 wherein the aryl group is phenyl.31. The method of claim 30 wherein phenyl is substituted with 1 or 2substituents selected from F, Cl, Br, I, OH, CH₃, CF₃, and CN.